Recombinant NEL-like (NELL) protein production

This application is a continuation of U.S. patent application Ser. No. 13/121,394; which is a national stage entry of International Application No. PCT/US09/59893 filed on Oct. 7, 2009; which in turn claims the benefit of U.S. Provisional Application No. 61/103,534 filed on Oct. 7, 2008; the teaching of each of which is incorporated by reference in its entirety.

The application also claims priority as a continuation-in-part to U.S. patent application Ser. No. 13/253,859; which is a division of U.S. patent application Ser. No. 12/700,630 filed on Feb. 4, 2010 and issued as U.S. Pat. No. 8,053,412; which in turn is a division of U.S. patent application Ser. No. 11/601,529 filed on Nov. 17, 2006 and issued as U.S. Pat. No. 7,691,607; which is a continuation-in-part of U.S. patent application Ser. No. 10/544,553 filed on May 15, 2006 and issued as U.S. Pat. No. 7,544,486; which is a continuation-in-part of International Application No. PCT/US2006/005473 filed on Feb. 16, 2006 and a national stage entry of International Application No. PCT/US2004/003808 filed on Feb. 9, 2004 the teaching of each of which is incorporated by reference in its entirety.

This invention was made with Government support under DE000422 and DE014649 awarded by the National Institutes of Health. The Government has certain rights in the invention.

The present invention is directed to a system and method of producing NELL peptides.

Growth factors are substances, such as peptides, which affect the growth and differentiation of defined populations of cells in vivo or in vitro.

Bone formation occurs during development of long bones (endochondral bone formation) and flat bones (intramembranous bone formation). Further, bone formation occurs during bone remodeling which occurs continuously in adult life in order to preserve the integrity of the skeleton. Finally, bone formation occurs during bone repair, such as when bone wounds occur in a fracture or surgical situation, for example. While separate bone formation mechanisms are thought to be involved in the embryological development of long and flat bones and repair is thought to involve intramembranous bone formation.

Bone formation by either mechanism involves the activity of osteoblasts, which are regulated by growth factors. Osteoblasts are derived from a pool of marrow stromal cells (also known as mesenchymal stem cells; MSC). These cells are present in a variety of tissues and are prevalent in bone marrow stroma. MSC are pluripotent and can differentiate into a variety of cell types including osteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes. Growth factors are thought to impact osteogenic cell proliferation, differentiation and osteoblast mineralization, each of which impacts bone formation.

Autogenous bone has been used, such to repair bone in patients with craniosynostosis and cleft grafting, for example. Craniosynostosis (CS), the premature closure of cranial sutures, affects 1 in 3,000 infants and therefore is one of the most common human congenital craniofacial deformities. Premature suture closure results in cranial dimorphism, which can need surgical correction. Premature suture closure in human CS can occur by two possibly distinct processes: calvarial overgrowth and bony fusion. Recently, fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) have been implicated in premature cranial suture fusion via CBFA1-mediated pathways (8). Missense mutation of CBFA1 is linked to cleidocranial dysplasia, manifested as delayed suture closure.

Autologous bone grafting procedures have been performed utilizing autogenous bone, such as from the iliac crest or calvaria. These donor sites are not without associated morbidity including pain, gait disturbance, thigh paresthesia for iliac crest donor sites, and infection, neurologic deficits, and hematomas for calvarial grafts. Further, donor sites can have limited volume and can contribute to increased surgical time and hospital stay.

Alloplastic grafting materials have also been utilized, and growth factors have been tested in animal models. For example, bFGF has shown potential for use in bone regeneration and repair. Another family of osteogenic growth factors have been described as bone morphogenic protein (BMP). Specifically, BMP-2 recombinant protein has been demonstrated to regenerate mandibular continuity defects and cleft palate defects with results equal to or better than autogenous particulate bone and marrow. BMPs and other osteogenic factors have been studied for use in clinical applications. However, the cost of using minimally effective dosages of BMP has been a limiting factor in clinical use.

Spinal fusion is a surgical technique in which one more of the vertebrae of the spine are united together so that motion no longer occurs between them. Indications include: treatment of a fractured (broken) vertebra, correction of deformity, elimination of pain from motion, treatment of instability, and treatment of some cervical disc herniations. The surgery can involve placement of a bone graft between the vertebrae to obtain a solid union between the vertebrae. The procedure also can involve supplemental treatments including the placement of plates, screws, cages, and recently bone morphogenic protein 2 and 7 to assist in stabilizing and healing the bone graft. Autogenous bone grafting has been the clinically preferred method, and yet has about a 30-50% failure rate. Autogenous bone grafting is a separate surgery and also carries significant morbidity.

Cartilage is a type of dense connective tissue. It is composed of chondrocytes which are dispersed in a firm gel-like matrix. Cartilage is avascular (contains no blood vessels) and nutrients are diffused through the matrix. Cartilage is found in the joints, the rib cage, the ear, the nose, in the throat and between intervertebral disks. There are three main types of cartilage: hyaline (e.g., costal cartilages, the cartilages of the nose, trachea, and bronchi, and the articular cartilages of joints), elastic (e.g., external ear, external auditory meatus, part of the Eustachian tube, epiglottis, and in some of the laryngeal cartilages) and fibrocartilage [e.g. meniscus (e.g., wrist triangular fibrocartilage complex, knee meniscus), intervertebral discs, temporomandibular joint disc, the pubic symphysis, and in some tendons and ligaments at their attachment to bones. One of the main purposes of cartilage is to provide a framework upon which bone deposition could begin (i.e., during endochondral ossification). Another important purpose of cartilage is to provide smooth surfaces for the movement of articulating bones. For example, articular cartilage, most notably that which is found in the knee joint, is generally characterized by very low friction, high wear resistance, and poor regenerative qualities. It is responsible for much of the compressive resistance and load bearing qualities of the knee joint and, without it, walking is painful to impossible. Yet another important purpose of cartilage is to provide, firm, yet flexible support (e.g., nasal cartilage, spinal discs, tracheal cartilage, knee meniscus, bronchial cartilage). For instance, cartilage such as the meniscus plays a crucial role in joint stability, lubrication, and force transmission. Under a weight bearing load, the meniscus maintains the balanced position of the femur on the tibia and distributes the compressive forces by increasing the surface contact area, thereby decreasing the average stress two to three times. Additionally, the menisci interact with the joint fluid to produce a coefficient of friction that is five times as slick as ice on ice. In another example, the intervertebral disc has several important functions, including functioning as a spacer, as a shock absorber, and as a motion unit. The gelatinous central portion of the disc is called the nucleus pulposus. It is composed of 80-90% water. The solid portion of the nucleus is Type II collagen and non-aggregated proteoglycans. The outer ligamentous ring around the nucleus pulposus is called the annulus fibrosus, which hydraulically seals the nucleus, and allows intradiscal pressures to rise as the disc is loaded. The annulus has overlapping radial bands, not unlike the plies of a radial tire, and this allows torsional stresses to be distributed through the annulus under normal loading without rupture. The disc functions as a hydraulic cylinder. The annulus interacts with the nucleus. As the nucleus is pressurized, the annular fibers serve a containment function to prevent the nucleus from bulging or herniating.

Cartilage can be damaged by wear, injury or diseases. As we age, the water and protein content of the body's cartilage changes. This change results in weaker, more fragile and thin cartilage. Osteoarthritis is a common condition of cartilage failure that can lead to limited range of motion, bone damage and invariably, pain. Due to a combination of acute stress and chronic fatigue, osteoarthritis directly manifests itself in a wearing away of the articulating surface and, in extreme cases, bone can be exposed in the joint. In another example, loss of the protective stabilizing meniscus leads to increased joint laxity or abnormal motions that lead to joint instability. The excessive motion and narrowed contact area promotes early arthritic changes. At the cellular level, there is initially a loss of cells from the superficial layer of the articular cartilage followed by cartilage splitting, subsequent thinning and erosion occurs, and finally protrusion of the underlying raw bone. The earliest arthritic changes have been noted three weeks after loss of the entire meniscus. In yet another example, because both the discs and the joints that stack the vertebrae (facet joints) are partly composed of cartilage, these areas are subject to wear and tear over time (degenerative changes). As the inner nucleus dehydrates, the disc space narrows, and redundant annular ligaments bulge. With progressive nuclear dehydration, the annular fibers can crack and tear. Loss of normal soft tissue tension may allow the spinal segment to sublux (e.g. partial dislocation of the joint), leading to osteophyte formation (bone spurs), foraminal narrowing, mechanical instability, and pain. If the annular fibers stretch or rupture, allowing the pressurized nuclear material to bulge or herniate and compress neural tissues, pain and weakness may result. This is the condition called a pinched nerve, slipped disc, or herniated disc. Radiculopathy refers to nerve irritation caused by damage to the disc between the vertebrae. Mechanical dysfunction may also cause disc degeneration and pain (e.g. degenerative disc disease). For example, the disc may be damaged as the result of some trauma that overloads the capacity of the disc to withstand increased forces passing through it, and inner or outer portions of the annular fibers may tear. These torn fibers may be the focus for inflammatory response when they are subjected to increased stress, and may cause pain directly, or through the compensatory protective spasm of the deep paraspinal muscles.

NELL peptide has been documented to stimulate bone formation and cartilage formation. Existing methods and systems of producing NELL peptides have limited success with problems. A common problem of existing NELL peptide expression systems (e.g., a mammalian cell such as a CHO cell expression system) is low productivity. Low productivity is generally found to be caused by, for example, an inappropriate construct of the nucleic acid sequence expressing a NELL peptide and/or other additional sequences, e.g., a secretory signal peptide sequence, in a particular expression system, e.g., a CHO cell.

Therefore there is a continuous need for a method and system for producing NELL peptides.

The present invention relates to a system and method for producing a NELL peptide. The method comprises:

providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a non-insect secretory signal peptide;

transfecting a mammalian cell with the nucleic acid construct; and

culturing the mammalian cell under conditions that permit expression of the NELL peptide.

The mammalian cell can be, e.g., a Chinese hamster ovary cell. The nucleic acid encodes NELL1 or NELL2 or both.

In some embodiments, the method further comprises: collecting NELL peptide secreted from the cell line; and substantially purifying the NELL peptide.

In some embodiments, the method of the above various embodiments can further comprise testing the activity of the NELL peptide to induce bone formation.

The act of purifying can be any step of purifying a protein or peptide. In some embodiments, the act of purifying includes chromatography purification.

In some embodiments, the present invention provides a nucleic acid construct for expressing a NELL peptide in a mammalian cell. The nucleic acid construct comprises at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a secretory signal peptide that is a non-insect secretory signal peptide. The nucleic acid encodes NELL1 or NELL2. In some embodiments, the mammalian cell can be a CHO cell.

In some embodiments, the present invention provides a mammalian cell line for expressing a NELL peptide. The cell line comprises a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a secretory signal peptide that is a non-insect secretory signal peptide. The nucleic acid encodes NELL1 or NELL2. In some embodiments, the mammalian cell can be a CHO cell.

In some embodiments, the present invention provides a polypeptide comprising a NELL peptide and a non-insect secretory signal peptide.

In some embodiments, the mammalian cell can be human embryo kidney cell, such as HEK-293.

The present invention provides a method of purifying a NELL peptide, comprising providing a crude solution containing a NELL peptide, and subjecting the crude solution to membrane chromotography to obtain a purified NELL peptide.

In some embodiments, the membrane chromatography is anion-exchange membrane chromatography or cation-exchange membrane chromatography.

The present invention provides a method of purifying a NELL peptide, comprising providing a crude solution containing a NELL peptide, and subjecting the crude solution to chromotography process to obtain a purified NELL peptide, wherein the chromatography process comprises a medium comprising a metal ion bearing two or more charges. In some embodiments, the metal ion is calcium ion.

The NELL peptide can be NELL-1 peptide or NELL-2 peptide.

FIGS. 1A-1D (SEQ ID NOs: 609 and 610) illustrate a signal peptide-NELL1-FLAG nucleic acid construct. The underlined amino acid sequence is from melittin signal peptide. The NELL1 peptide is from rat NELL1 peptide.

FIG. 2A is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide.

FIG. 2B shows a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression in reference to a protein ladder.

FIG. 2C is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing NELL1-FLAG.

FIG. 2D shows a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression.

FIG. 3A depicts the nucleic acid sequence of the cDNA construct and amino acid sequences of three different signal peptides (SEQ ID NOs: 611-613) that were used for the constructs.

FIG. 3B is a Western blot with anti-c-myc antibody detecting secreting NELL1 from transfections with different constructs after immunoprecipitation using anti-c-myc agarose.

FIG. 3C is a Western blot with anti-c-myc or mouse anti-human NELL1 antibodies detecting secreting NELL1 after immunoprecipitation using rabbit anti-human Nell-1 antibody-NHS activated sepharose.

FIGS. 4A-D show NELL1 ( homo sapiens ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 1 and 2).

FIGS. 5A-D show NELL1 ( rattus norvegicus ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 3 and 4).

FIGS. 6A-F show NELL1 ( Mus musculs ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 5 and 6).

FIGS. 7A-D show NELL2 ( homo sapiens ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 7 and 8).

FIGS. 8A-D show NELL2 ( rattus norvegicus ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 9 and 10).

FIGS. 9A-D show NELL2 ( Mus musculs ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 11 and 12).

FIGS. 10A-D show NELL2 ( Gallus gallus ) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 13 and 14).

As used herein, the terms “polypeptide”, “peptide” and “protein” can be used interchangeably to refer to a polymer of amino acid residues. The terms can apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term “antibody” can include various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond, a Fab or F(ab′) 2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like. An antibody can include intact molecules as well as fragments thereof, such as, Fab and F(ab′) 2 , and/or single-chain antibodies (e.g. scFv) which can bind an epitopic determinant. An antibody can be of animal, e.g., mouse or rat, or of human origin, or can be chimeric or humanized. Antibodies can be polyclonal or monoclonal antibodies (mAbs), such as monoclonal antibodies with specificity for a polypeptide encoded by a NELL1 or NELL 2 protein.

The term “capture agent” refers to molecules that specifically bind other molecules to form a binding complex such as, but not limited to, an antibody-antigen complex, a lectin-carbohydrate complex, a nucleic acid-nucleic acid complex or a biotin-avidin complex.

The term “specifically binds” refers to the binding property of a biomolecule (e.g., protein, nucleic acid, antibody, etc.), which is determinative of the presence of such a biomolecule in a heterogeneous population of molecules (e.g., proteins and other biologics). Thus, under designated conditions (e.g., immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody specifically binds to its particular “target” molecule and will not bind in a significant amount to other molecules present in the sample.

The terms “nucleic acid” or “oligonucleotide” refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention can be single-stranded or double stranded and can contain phosphodiester bonds, although in some cases, nucleic acid analogs can be included that can have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, omethylphophoroamidite linkages, and/or peptide nucleic acid backbones and linkages. Analog nucleic acids can have positive backbones and/or non-ribose backbones. Nucleic acids can also include one or more carbocyclic sugars. Modifications of the ribose-phosphate backbone can be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments, for example.

The term “specific hybridization” refers to the preferential binding, duplexing, or hybridizing of a nucleic acid molecule to a particular nucleotide sequence under stringent conditions, including conditions under which a probe can hybridize preferentially to its target subsequence, and can hybridize to a lesser extent to other sequences.

The terms “NELL1 cDNA” refer to SEQ ID NO:1, 3 and 5, and “NELL2 cDNA” can refer to SEQ ID NO:7, 9, 11 and 13.

NELL Peptides

NELL1 is a 810 aa (amino acid) peptide, distributed primarily in bone. In adults, NELL1 is expressed at high levels in craniofacial bone, and lower levels in long bone. NELL1 has known roles in osteoblast differentiation, bone formation and bone regeneration. NELL1 has known rules in forming cartilage tissues without forming bone.

NELL 2 is a 816 aa peptide, distributed in neural cells and brain.

The human NELL1 gene includes at least 3 Cbfa1 response elements in the promoter region. Cbfa1 specifically binds to these response elements. NELL1 expression can be under the control of these transcription factors expressed endogenously at least in preosteoblasts, osteoblasts and hypertrophic chondrocytes in development and in adulthood. Cleidocranial disostosis is a developmental cranial defect thought to be caused at least in part by Cbfa disruption.

A NELL1 peptide can be encoded by the NELL1 gene or cDNA and includes SEQ ID NO: 2, 4, and 6. The NELL1 peptide can include a NELL1 peptide fragment that retains the ability to induce osteogenic cell differentiation, osteoblast differentiation or bone formation. In some embodiments, the NELL1 peptide can include a NELL 1 peptide fragment that retains the ability to induce cartilage formation without forming bone. A NELL2 peptide can be encoded by the NELL2 gene or cDNA and includes SEQ ID NO: 8, 10, 12 and 14. The NELL2 peptide can include NELL2 peptide fragments that retain similar activity to the full NELL2 peptide sequence.

In some embodiments, the amino acid sequence of the NELL peptide can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:2), rat NELL1 (SEQ ID NO:4), mouse NELL1 (SEQ ID NO:6), or human NELL2 (SEQ ID NO:8), rat NELL2 (SEQ ID NO:10), mouse NELL2 (SEQ ID NO:12), chicken NELL2 (SEQ ID NO:14). The amino acid sequence can also include sequences such as those with substantial similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above, or contain similar active binding domains as NELL1 peptides.

The term “derivative” as used herein, refers to any chemical or biological compound or material derived from a NELL peptide, structural equivalents thereof or conformational equivalents thereof. For example, such a derivative can include any pro-drug form, PEGylated form, or any other form of a NELL peptide that renders the NELL peptide more stable or more osteophilic or lipophilic. In some embodiments, the derivative can be a NELL peptide attached to poly(ethylene glycol), a poly(amino acid), a hydrocarbyl short chain having C1-C20 carbons, or a biocompatible polymer. In some embodiments, the term “derivative” can include NELL peptide mimetics. As used herein, the term “mimetic” refers to a peptide having at least one non-peptide bond in its backbone. A peptide bond is a chemical bond formed between the carboxylic acid group of an amino acid molecule and the amino group of another amino acid molecule. A NELL peptide mimetic can be any compound that exhibits at least one or more bone forming, bone repairing, cartilage forming, and/or cartilage repairing function of a NELL peptide.

Synthetic methods for making peptide mimetics are well known in the art. The following describes an example of the basic procedure for the synthesis of a peptide, including a peptide mimetics:

Before the peptide synthesis starts, the amine terminus of the amino acid (starting material) can protected with FMOC (9-fluoromethyl carbamate) or other protective groups, and a solid support such as a Merrifield resin (free amines) is used as an initiator. Then, step (1) through step (3) reactions are performed and repeated until the desired peptide is obtained: (1) a free-amine is reacted with carboxyl terminus using carbodiimide chemistry, (2) the amino acid sequence is purified, and (3) the protecting group, e.g., the FMOC protecting group, is removed under mildly acidic conditions to yield a free amine. The peptide can then be cleaved from the resin to yield a free standing peptide or peptide mimetics.

Systems Expressing NELL Peptides

Generally, the method of invention includes providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding a non-insect signal peptide.

In one embodiment, the method can include transfecting an insect cell line with a nucleic acid construct encoding a NELL peptide; and culturing the insect cell line under conditions that permit expression and/or secretion of the NELL peptide. For example, the cell line can be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide.

In one embodiment, the method can include providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. The nucleic acid sequence can be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). In some embodiments, the nucleic acid sequence can also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

Further the nucleic acid can include an expression vector for expressing the nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. For example, the expression vector can be pIZT/V5-His (Invitrogen), and selective markers can also include blastcidin and neomycin.

Such expression systems can include a carrier such as a viral carrier or viral vector, peptide carrier or a short polymer molecule.

Nucleic acid constructs can comprise expression and cloning vectors should contain a selection gene, also termed a selectable marker, such as a gene that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector will not obtain an advantage in growth or reproduction over transformed hosts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies.

Further, the nucleic acid sequence can also include additional nucleic acids which encode reporter products to monitor levels of gene expression, or encode peptide tags which can be visualized using known methods in the art to monitor levels of peptide expression. Additional sequences can be selected so as to not interfere with the expression of the nucleic acid, or the functionality of the expressed peptide product.

A promoter is recognized by the host organism and is operatively linked to the NELL encoding nucleic acid. Promoters are untranslated sequences located upstream from the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control, including inducible and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known.

A nucleic acid can be operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to facilitate translation.

A NELL peptide can be expressed in any biological system. For example, a NELL peptide can be expressed in a bacterial system, a yeast system, a plant system or animal system.

In some embodiments, a NELL peptide can be expressed in a cell free expression system well known to those in the art. For example, E coli cell-free protein translation systems or wheat germ cell-free protein translation systems.

In some embodiments, a NELL peptide can be expressed in transgenic plant cell systems derived from tobacco, corn, rice or soybean.

In some embodiments, a NELL peptide can be expressed in insect cells. The NELL1 and NELL2 peptides expressed in an insect system are functional forms of the protein.

COS7 cells can be used to produce NELL1 and NELL2 proteins at low levels, such as about 10 micrograms per litter medium, but require serum-containing medium for the expression. As for the signal peptides, NELL1 and NELL2 endogenous signal peptides permit expression in COS7 cells.

In one embodiment, the invention includes a method of expressing a functional NELL peptide, such as NELL1 or NELL2 peptide, using an insect cell line. In one embodiment, the insect cell can be a high five cell, Sf9 and other Sf cells.

In one embodiment, the invention can include a nucleic acid construct for expressing a NELL peptide, such as NELL1 and/or NELL2 peptide in an insect cell. The nucleic acid sequence can be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). The nucleic acid sequence can also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

In one embodiment, the invention can include a nucleic acid construct for expressing a NELL peptide, such as NELL1 and/or NELL2 peptide in a mammalian cell such as a Chinese hamster ovary cell (CHO cell). The nucleic acid sequence can be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). In some embodiments, the nucleic acid sequence can also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

In one embodiment, the invention can include cells that express functional NELL peptides. For example, the cell can be a CHO cell. In one embodiment, the cell can be transfected with a nucleic acid construct encoding a NELL peptide. For example, the cell line can be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide. In one embodiment, NELL expressing nucleic acids (e.g., cDNA(s) can be cloned into gene expression vector or viral particles that are competent to transfect cells (such as insect cells or Chinese hamster ovary cells (CHO cells)).

The nucleic acid construct can also include a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding a non-insect signal peptide that is a non-insect secretory signal peptide.

In one embodiment, the invention can include cells that express functional NELL peptides, and can secrete functional proteins.

In one embodiment, the invention can include a polypeptide (amino acid sequence) comprising a NELL peptide, such as NELL1 or NELL2 peptide, and can include a non-insect secretory signal peptide.

Non-Insect Secretory Signal Peptide

Useful non-insect secretory signal peptide for use in a NELL peptide expression system (e.g., a mammalian cell such as a CHO cell) can be any secretory signal peptide. Such non-insect secretory signal peptide can be, for example, plant secretory signal peptide, or animal secretory signal peptide, e.g., mammalian secretory signal peptide such as human secretory signal peptide. Various secretory signal peptide can be found at various publicly accessible sources, e.g., http<://>proline<dot>bic<dot>nus<dot>edu<dot>sg.

Human secretory signal peptides are well documented. Examples of human secretory signal peptides include, but are not limited to,

1A02_human secretory signal peptide, 1A11_human secretory signal peptide, 1A25_human secretory signal peptide, 1A26_human secretory signal peptide, 1A30_human secretory signal peptide, 1A31_human secretory signal peptide, 1A33_human secretory signal peptide, 1A68_human secretory signal peptide, 1A80_human secretory signal peptide, 1B07_human secretory signal peptide, 1B15_human secretory signal peptide, 1B37_human secretory signal peptide, 1B40_human secretory signal peptide, 1B47_human secretory signal peptide, 1B48_human secretory signal peptide, 1B57_human secretory signal peptide, 1B59_human secretory signal peptide, 1B78_human secretory signal peptide, 1C03_human secretory signal peptide, 1C04_human secretory signal peptide, 1C05_human secretory signal peptide, 1C07_human secretory signal peptide, 1C14_human secretory signal peptide, 1C15_human secretory signal peptide, 1C16_human secretory signal peptide, 1C17_human secretory signal peptide, 1C18_human secretory signal peptide, 2B11_human secretory signal peptide, 2B14_human secretory signal peptide, 2B17_human secretory signal peptide, 2B1A_human secretory signal peptide, 2B1B_human secretory signal peptide, 2B32_human secretory signal peptide, 2DOB_human secretory signal peptide, 7B2_human secretory signal peptide, A1AT_human secretory signal peptide, A2GL_human secretory signal peptide, A2MG_human secretory signal peptide, ABP1_human secretory signal peptide, ACET_human secretory signal peptide, ACHB_human secretory signal peptide, ACHE_human secretory signal peptide, ACRO_human secretory signal peptide, ADA32_human secretory signal peptide, ADIPO_human secretory signal peptide, ADML_human secretory signal peptide, AGAL_human secretory signal peptide, AGR2_human secretory signal peptide, AGR3_human secretory signal peptide, AMBP_human secretory signal peptide, AMTN_human secretory signal peptide, ANF_human secretory signal peptide, ANGI_human secretory signal peptide, ANGL3_human secretory signal peptide, ANGL7_human secretory signal peptide, ANGT_human secretory signal peptide, ANPRA_human secretory signal peptide, APOA2_human secretory signal peptide, APOA4_human secretory signal peptide, APOA_human secretory signal peptide, APOC1_human secretory signal peptide, APOC2_human secretory signal peptide, APOC3_human secretory signal peptide, APOD_human secretory signal peptide, APOE_human secretory signal peptide, APOH_human secretory signal peptide, APR3_human secretory signal peptide, ASM_human secretory signal peptide, ASPG_human secretory signal peptide, BAMBI_human secretory signal peptide, BASI_human secretory signal peptide, BGAL_human secretory signal peptide, BGLR_human secretory signal peptide, BOC_human secretory signal peptide, BPIL1_human secretory signal peptide, BPI_human secretory signal peptide, BT3A 3 _human secretory signal peptide, BTNL8_human secretory signal peptide, C16L2_human secretory signal peptide, C1QT5_human secretory signal peptide, C1QT6_human secretory signal peptide, C1R_human secretory signal peptide, C1S_human secretory signal peptide, C4BPA_human secretory signal peptide, CA187_human secretory signal peptide, CADM3_human secretory signal peptide, CAH9_human secretory signal peptide, CALCR_human secretory signal peptide, CALRL_human secretory signal peptide, CALR_human secretory signal peptide, CAP7_human secretory signal peptide, CART_human secretory signal peptide, CASA1_human secretory signal peptide, CASB_human secretory signal peptide, CASK_human secretory signal peptide, CATC_human secretory signal peptide, CATE_human secretory signal peptide, CATG_human secretory signal peptide, CATW_human secretory signal peptide, CBG_human secretory signal peptide, CBLN3_human secretory signal peptide, CBLN4_human secretory signal peptide, CBPA1_human secretory signal peptide, CBPA3_human secretory signal peptide, CBPB1_human secretory signal peptide, CBPN_human secretory signal peptide, CCL11_human secretory signal peptide, CCL15_human secretory signal peptide, CCL19_human secretory signal peptide, CCL1_human secretory signal peptide, CCL22_human secretory signal peptide, CCL24_human secretory signal peptide, CCL2_human secretory signal peptide, CCL4_human secretory signal peptide, CCL5_human secretory signal peptide, CCL7_human secretory signal peptide, CD180_human secretory signal peptide, CD1A_human secretory signal peptide, CD244_human secretory signal peptide, CD276_human secretory signal peptide, CD27_human secretory signal peptide, CD28_human secretory signal peptide, CD2_human secretory signal peptide, CD320_human secretory signal peptide, CD34_human secretory signal peptide, CD3E_human secretory signal peptide, CD3G_human secretory signal peptide, CD3Z_human secretory signal peptide, CD45_human secretory signal peptide, CD5L_human secretory signal peptide, CD5_human secretory signal peptide, CD83_human secretory signal peptide, CD8B_human secretory signal peptide, CD99_human secretory signal peptide, CEAM1_human secretory signal peptide, CER1_human secretory signal peptide, CERU_human secretory signal peptide, CETP_human secretory signal peptide, CF126_human secretory signal peptide, CFAB_human secretory signal peptide, CFAH_human secretory signal peptide, CFAI_human secretory signal peptide, CH3L1_human secretory signal peptide, CH3L2_human secretory signal peptide, CHIT1_human secretory signal peptide, CL3L1_human secretory signal peptide, CLC11_human secretory signal peptide, CLC14_human secretory signal peptide, CLM1_human secretory signal peptide, CLM9_human secretory signal peptide, CLUS_human secretory signal peptide, CMA1_human secretory signal peptide, CMGA_human secretory signal peptide, CO1A1_human secretory signal peptide, CO2_human secretory signal peptide, CO3_human secretory signal peptide, CO4A2_human secretory signal peptide, CO5A2_human secretory signal peptide, CO6A1_human secretory signal peptide, CO6_human secretory signal peptide, CO7_human secretory signal peptide, CO9A1_human secretory signal peptide, CO9_human secretory signal peptide, COGA1_human secretory signal peptide, COLI_human secretory signal peptide, COL_human secretory signal peptide, CR2_human secretory signal peptide, CRDL2_human secretory signal peptide, CREG1_human secretory signal peptide, CRHBP_human secretory signal peptide, CRIM1_human secretory signal peptide, CRIS1_human secretory signal peptide, CRLF1_human secretory signal peptide, CRP_human secretory signal peptide, CSF2R_human secretory signal peptide, CSF2_human secretory signal peptide, CSF3R_human secretory signal peptide, CSPG2_human secretory signal peptide, CST9L_human secretory signal peptide, CST9_human secretory signal peptide, CTHR1_human secretory signal peptide, CTRB1_human secretory signal peptide, CXCL7_human secretory signal peptide, CXL10_human secretory signal peptide, CYTC_human secretory signal peptide, CYTL1_human secretory signal peptide, CYTN_human secretory signal peptide, CYTS_human secretory signal peptide, CYTT_human secretory signal peptide, D103A_human secretory signal peptide, DB127_human secretory signal peptide, DCD_human secretory signal peptide, DEF1_human secretory signal peptide, DKK1_human secretory signal peptide, DKK3_human secretory signal peptide, DKK4_human secretory signal peptide, DLK_human secretory signal peptide, DLL4_human secretory signal peptide, DNAS1_human secretory signal peptide, ECP_human secretory signal peptide, EDAR_human secretory signal peptide, EFNB1_human secretory signal peptide, EFNB3_human secretory signal peptide, EGFL8_human secretory signal peptide, EGFR_human secretory signal peptide, EGLN_human secretory signal peptide, ELA2A_human secretory signal peptide, ELA2B_human secretory signal peptide, ELAF_human secretory signal peptide, ENPL_human secretory signal peptide, ENPP7_human secretory signal peptide, EPGN_human secretory signal peptide, EPHB1_human secretory signal peptide, EPHB6_human secretory signal peptide, EPOR_human secretory signal peptide, EPO_human secretory signal peptide, ESAM_human secretory signal peptide, EST1_human secretory signal peptide, F13B_human secretory signal peptide, FA11_human secretory signal peptide, FA5_human secretory signal peptide, FA8_human secretory signal peptide, FCGR1_human secretory signal peptide, FCGRN_human secretory signal peptide, FCN1_human secretory signal peptide, FCN3_human secretory signal peptide, FCRL2_human secretory signal peptide, FCRLA_human secretory signal peptide, FETUA_human secretory signal peptide, FGF19_human secretory signal peptide, FGF21_human secretory signal peptide, FGF23_human secretory signal peptide, FGFR3_human secretory signal peptide, FGFR4_human secretory signal peptide, FGRL1_human secretory signal peptide, FIBA_human secretory signal peptide, FIBG_human secretory signal peptide, FKB14_human secretory signal peptide, FKBP2_human secretory signal peptide, FLRT2_human secretory signal peptide, FSHB_human secretory signal peptide, FSTL1_human secretory signal peptide, FSTL3_human secretory signal peptide, FST_human secretory signal peptide, FZD3_human secretory signal peptide, G6B_human secretory signal peptide, GALC_human secretory signal peptide, GDN_human secretory signal peptide, GELS_human secretory signal peptide, GI24_human secretory signal peptide, GLHA_human secretory signal peptide, GLPA_human secretory signal peptide, GLPB_human secretory signal peptide, GLPE_human secretory signal peptide, GLUC_human secretory signal peptide, GNS_human secretory signal peptide, GP1BA_human secretory signal peptide, GPI8_human secretory signal peptide, GPIX_human secretory signal peptide, GPR56_human secretory signal peptide, GPR97_human secretory signal peptide, GRAB human secretory signal peptide, GREM1_human secretory signal peptide, GROA_human secretory signal peptide, GRP78_human secretory signal peptide, GRP_human secretory signal peptide, HA22_human secretory signal peptide, HA23_human secretory signal peptide, HA25_human secretory signal peptide, HA27_human secretory signal peptide, HB21_human secretory signal peptide, HB23_human secretory signal peptide, HB24_human secretory signal peptide, HB25_human secretory signal peptide, HB2B_human secretory signal peptide, HB2C_human secretory signal peptide, HB2K_human secretory signal peptide, HEP2_human secretory signal peptide, HGFA_human secretory signal peptide, HIS1_human secretory signal peptide, HIS3_human secretory signal peptide, HLAE_human secretory signal peptide, HPSE_human secretory signal peptide, HPT_human secretory signal peptide, HV103_human secretory signal peptide, HV303_human secretory signal peptide, I10R1_human secretory signal peptide, I10R2_human secretory signal peptide, I17RA_human secretory signal peptide, I17RB_human secretory signal peptide, I17RC_human secretory signal, I18BP_human secretory signal peptide, I20RA_human secretory signal peptide, I20RB_human secretory signal peptide, I22RA_human secretory signal peptide, IBP1_human secretory signal peptide, IBP2_human secretory signal peptide, IBP3_human secretory signal peptide, IBP7_human secretory signal peptide, IFN16_human secretory signal peptide, IFNA2_human secretory signal peptide, IFNA4_human secretory signal peptide, IFNA5_human secretory signal peptide, IFNA6_human secretory signal peptide, IFNA7_human secretory signal peptide, IFNK_human secretory signal peptide, IFNW1_human secretory signal peptide, IGF2_human secretory signal peptide, IGFL1_human secretory signal peptide, IGFL3_human secretory signal peptide, IL10_human secretory signal peptide, IL12A_human secretory signal peptide, IL12B_human secretory signal peptide, IL17F_human secretory signal peptide, IL17_human secretory signal peptide, IL19_human secretory signal peptide, IL1R1_human secretory signal peptide, IL1RA_human secretory signal peptide, IL20_human secretory signal peptide, IL21R_human secretory signal peptide, IL22_human secretory signal peptide, IL24_human secretory signal peptide, IL25_human secretory signal peptide, IL2RA_human secretory signal peptide, IL2RB_human secretory signal peptide, IL2RG_human secretory signal peptide, IL2_human secretory signal peptide, IL3_human secretory signal peptide, IL4_human secretory signal peptide, IL5RA_human secretory signal peptide, IL6RA_human secretory signal peptide, IL7RA_human secretory signal peptide, IL7_human secretory signal peptide, IL9_human secretory signal peptide, ILRL1_human secretory signal peptide, INAR2_human secretory signal peptide, INHA_human secretory signal peptide, INSL3_human secretory signal peptide, INSL4_human secretory signal peptide, INSL5_human secretory signal peptide, INS_human secretory signal peptide, IPSP_human secretory signal peptide, IRBP_human secretory signal peptide, ISK6_human secretory signal peptide, ITA2B_human secretory signal peptide, ITA2_human secretory signal peptide, ITA3_human secretory signal peptide, ITA4_human secretory signal peptide, ITA6_human secretory signal peptide, ITA7_human secretory signal peptide, ITAE_human secretory signal peptide, ITAL_human secretory signal peptide, ITAV_human secretory signal peptide, ITAX_human secretory signal peptide, ITB2_human secretory signal peptide, ITB4_human secretory signal peptide, ITIH4_human secretory signal peptide, JAM1_human secretory signal peptide, JAM2_human secretory signal peptide, JAM3_human secretory signal peptide, JAML1_human secretory signal peptide, KAZD1_human secretory signal peptide, KI2L1_human secretory signal peptide, KIRR2_human secretory signal peptide, KLK3_human secretory signal peptide, KLKB1_human secretory signal peptide, KNG1_human secretory signal peptide, KTEL1_human secretory signal peptide, KV403_human secretory signal peptide, KV404_human secretory signal peptide, L1CAM_human secretory signal peptide, LALBA_human secretory signal peptide, LAMB1_human secretory signal peptide, LAMC1_human secretory signal peptide, LAMP1_human secretory signal peptide, LAMP2_human secretory signal peptide, LBP_human secretory signal peptide, LCAT_human secretory signal peptide, LCN1_human secretory signal peptide, LCTL_human secretory signal peptide, LEUK_human secretory signal peptide, LG3BP_human secretory signal peptide, LIF_human secretory signal peptide, LIPG_human secretory signal peptide, LIPP_human secretory signal peptide, LIRA3_human secretory signal peptide, LMAN1_human secretory signal peptide, LPH_human secretory signal peptide, LRC55_human secretory signal peptide, LRRN1_human secretory signal peptide, LSHB_human secretory signal peptide, LUM_human secretory signal peptide, LU_human secretory signal peptide, LV605_human secretory signal peptide, LY86_human secretory signal peptide, LYAM2_human secretory signal peptide, LYPA3_human secretory signal peptide, LYPD6_human secretory signal peptide, LYSC_human secretory signal peptide, MBL2_human secretory signal peptide, MCP_human secretory signal peptide, MFAP4_human secretory signal peptide, MGP_human secretory signal peptide, MIA human secretory signal peptide, MIME_human secretory signal peptide, MIP2A_human secretory signal peptide, MIP2B_human secretory signal peptide, MK human secretory signal peptide, MMP1_human secretory signal peptide, MOTI_human secretory signal peptide, MOX2R_human secretory signal peptide, MPRD_human secretory signal peptide, MPRI_human secretory signal peptide, MPZL3_human secretory signal peptide, MSMB_human secretory signal peptide, MYPO_human secretory signal peptide, NAGAB_human secretory signal peptide, NELL1_human secretory signal peptide, NELL2_human secretory signal peptide, NETO2_human secretory signal peptide, NEU1_human secretory signal peptide, NEU2_human secretory signal peptide, NGAL_human secretory signal peptide, NID1_human secretory signal peptide, NID2_human secretory signal peptide, NLGNX_human secretory signal peptide, NMB_human secretory signal peptide, NOV_human secretory signal peptide, NPTN_human secretory signal peptide, NPY_human secretory signal peptide, NRP1_human secretory signal peptide, NTRK2_human secretory signal peptide, NXPH3_human secretory signal peptide, OLFL1_human secretory signal peptide, OTOR_human secretory signal peptide, OXLA_human secretory signal peptide, P3IP 1_human secretory signal peptide, PAHO_human secretory signal peptide, PARM1_human secretory signal peptide, PCDBA_human secretory signal peptide, PCOC2_human secretory signal peptide, PCYXL_human secretory signal peptide, PDGFA_human secretory signal peptide, PDIA1_human secretory signal peptide, PDIA3_human secretory signal peptide, PDYN_human secretory signal peptide, PEBP4_human secretory signal peptide, PECA1_human secretory signal peptide, PG12B_human secretory signal peptide, PGFRA_human secretory signal peptide, PGFRB_human secretory signal peptide, PGH1_human secretory signal peptide, PGRP2_human secretory signal peptide, PIGT_human secretory signal peptide, PIP_human secretory signal peptide, PLBL2_human secretory signal peptide, PLF4_human secretory signal peptide, PLOD1_human secretory signal peptide, PORIM_human secretory signal peptide, PPA6_human secretory signal peptide, PPAP_human secretory signal peptide, PPGB_human secretory signal peptide, PPIB_human secretory signal peptide, PRB4_human secretory signal peptide, PRLR_human secretory signal peptide, PRL_human secretory signal peptide, PROK1_human secretory signal peptide, PROK2_human secretory signal peptide, PROP_human secretory signal peptide, PROZ_human secretory signal peptide, PRP1_human secretory signal peptide, PRPC_human secretory signal peptide, PRRT3_human secretory signal peptide, PTGDS_human secretory signal peptide, PTHY_human secretory signal peptide, PTPRG_human secretory signal peptide, PYY_human secretory signal peptide, PZP_human secretory signal peptide, REG1A_human secretory signal peptide, REG3G_human secretory signal peptide, RIB1_human secretory signal peptide, RIB2_human secretory signal peptide, RISC_human secretory signal peptide, RNAS1_human secretory signal peptide, RNAS4_human secretory signal peptide, S39A6_human secretory signal peptide, SAA4_human secretory signal peptide, SAA_human secretory signal peptide, SAMP_human secretory signal peptide, SCG1_human secretory signal peptide, SCRG1_human secretory signal peptide, SEM4B_human secretory signal peptide, SEM6B_human secretory signal peptide, SEMG1_human secretory signal peptide, SEMG2_human secretory signal peptide, SEPP1_human secretory signal peptide, SFRP2_human secretory signal peptide, SFRP3_human secretory signal peptide, SFTPG_human secretory signal peptide, SG1D4_human secretory signal peptide, SG3A1_human secretory signal peptide, SHBG_human secretory signal peptide, SIAL_human secretory signal peptide, SIDT2_human secretory signal peptide, SLAF6_human secretory signal peptide, SLAF7_human secretory signal peptide, SLAF8_human secretory signal peptide, SLPI_human secretory signal peptide, SMR3B_human secretory signal peptide, SMS_human secretory signal peptide, SODS_human secretory signal peptide, SOSD1_human secretory signal peptide, SOST_human secretory signal peptide, SPIT1_human secretory signal peptide, SPIT2_human secretory signal peptide, SRCH_human secretory signal peptide, SRGN_human secretory signal peptide, STAT_human secretory signal peptide, STC1_human secretory signal peptide, TCO1_human secretory signal peptide, TCO2_human secretory signal peptide, TENA_human secretory signal peptide, TETN_human secretory signal peptide, TFF1_human secretory signal peptide, TFF3_human secretory signal peptide, TFPI1_human secretory signal peptide, TGFR2_human secretory signal peptide, THBG_human secretory signal peptide, THYG_human secretory signal peptide, TICN2_human secretory signal peptide, TIE1_human secretory signal peptide, TIE2_human secretory signal peptide, TIMP1_human secretory signal peptide, TIMP2_human secretory signal peptide, TIMP3_human secretory signal peptide, TINAL_human secretory signal peptide, TLR1_human secretory signal peptide, TLR3_human secretory signal peptide, TLR4_human secretory signal peptide, TLR5_human secretory signal peptide, TM2D1_human secretory signal peptide, TMIG2_human secretory signal peptide, TMM25_human secretory signal peptide, TMM46_human secretory signal peptide, TMM66_human secretory signal peptide, TMM9B_human secretory signal peptide, TNFB_human secretory signal peptide, TNR14_human secretory signal peptide, TNR16_human secretory signal peptide, TNR18_human secretory signal peptide, TNR19_human secretory signal peptide, TNR1B_human secretory signal peptide, TNR5_human secretory signal peptide, TNR6B_human secretory signal peptide, TNR8_human secretory signal peptide, TNR9_human secretory signal peptide, TPO_human secretory signal peptide, TPSNR_human secretory signal peptide, TPSN_human secretory signal peptide, TR10D_human secretory signal peptide, TR11B_human secretory signal peptide, TR19L_human secretory signal peptide, TRBM_human secretory signal peptide, TRFE_human secretory signal peptide, TRFL_human secretory signal peptide, TRY1_human secretory signal peptide, TRY2_human secretory signal peptide, TSHB_human secretory signal peptide, TSP1_human secretory signal peptide, TVA2_human secretory signal peptide, TVB2_human secretory signal peptide, TXD12_human secretory signal peptide, TXND4_human secretory signal peptide, TYRP1_human secretory signal peptide, UROK_human secretory signal peptide, UTER_human secretory signal peptide, UTS2_human secretory signal peptide, VCAM1_human secretory signal peptide, VEGFA_human secretory signal peptide, VEGFC_human secretory signal peptide, VGFR3_human secretory signal peptide, VMO1_human secretory signal peptide, VSIG2_human secretory signal peptide, VSIG4_human secretory signal peptide, VSTM1_human secretory signal peptide, VTNC_human secretory signal peptide, VWF_human secretory signal peptide, WISP2_human secretory signal peptide, X3CL1_human secretory signal peptide, XCL2_human secretory signal peptide, YK001_human secretory signal peptide, YQ001_human secretory signal peptide, ZA2G_human secretory signal peptide, ZG16_human secretory signal peptide, ZP2_human secretory signal peptide.

The sequences of these secretory signal peptides are as following:

1A02_HUMAN SPdb195  Homo sapiens  (Human)

(SEQ ID NO: 15)

1A11_HUMAN SPdb208  Homo sapiens  (Human)

(SEQ ID NO: 16)

1A25_HUMAN SPdb276  Homo sapiens  (Human)

(SEQ ID NO: 17)

1A26_HUMAN SPdb277  Homo sapiens  (Human)

(SEQ ID NO: 18)

1A30_HUMAN SPdb279  Homo sapiens  (Human)

(SEQ ID NO: 19)

1A31_HUMAN SPdb280  Homo sapiens  (Human)

(SEQ ID NO: 20)

1A33_HUMAN SPdb282  Homo sapiens  (Human)

(SEQ ID NO: 21)

1A68_HUMAN SPdb287  Homo sapiens  (Human)

(SEQ ID NO: 22)

1A80_HUMAN SPdb290  Homo sapiens  (Human)

(SEQ ID NO: 23)

1B07_HUMAN SPdb298  Homo sapiens  (Human)

(SEQ ID NO: 24)

1B15_HUMAN SPdb302  Homo sapiens  (Human)

(SEQ ID NO: 25)

1B37_HUMAN SPdb306  Homo sapiens  (Human)

(SEQ ID NO: 26)

1B40_HUMAN SPdb309  Homo sapiens  (Human)

(SEQ ID NO: 27)

1B47_HUMAN SPdb315  Homo sapiens  (Human)

(SEQ ID NO: 28)

1B48_HUMAN SPdb316  Homo sapiens  (Human)

(SEQ ID NO: 29)

1B57_HUMAN SPdb325  Homo sapiens  (Human)

(SEQ ID NO: 30)

1B59_HUMAN SPdb327  Homo sapiens  (Human)

(SEQ ID NO: 31)

1B78_HUMAN SPdb330  Homo sapiens  (Human)

(SEQ ID NO: 32)

1C03_HUMAN SPdb340  Homo sapiens  (Human)

(SEQ ID NO: 33)

1C04_HUMAN SPdb342  Homo sapiens  (Human)

(SEQ ID NO: 34)

1C05_HUMAN SPdb343  Homo sapiens  (Human)

(SEQ ID NO: 35)

1C07_HUMAN SPdb345  Homo sapiens  (Human)

(SEQ ID NO: 36)

1C14_HUMAN SPdb348  Homo sapiens  (Human)

(SEQ ID NO: 37)

1C15_HUMAN SPdb349  Homo sapiens  (Human)

(SEQ ID NO: 38)

1C16_HUMAN SPdb350  Homo sapiens  (Human)

(SEQ ID NO: 39)

1C17_HUMAN SPdb351  Homo sapiens  (Human)

(SEQ ID NO: 40)

1C18_HUMAN SPdb352  Homo sapiens  (Human)

(SEQ ID NO: 41)

2B11_HUMAN SPdb440  Homo sapiens  (Human)

(SEQ ID NO: 42)

2B14_HUMAN SPdb441  Homo sapiens  (Human)

(SEQ ID NO: 43)

2B17_HUMAN SPdb442  Homo sapiens  (Human)

(SEQ ID NO: 44)

2B1A_HUMAN SPdb445  Homo sapiens  (Human)

(SEQ ID NO: 45)

2B1B_HUMAN SPdb446  Homo sapiens  (Human)

(SEQ ID NO: 46)

2B32_HUMAN SPdb449  Homo sapiens  (Human)

(SEQ ID NO: 47)

2DOB_HUMAN SPdb458  Homo sapiens  (Human)

(SEQ ID NO: 48)

7B2_HUMAN SPdb1113  Homo sapiens  (Human)

(SEQ ID NO: 49)

A1AT_HUMAN SPdb1182  Homo sapiens  (Human)

(SEQ ID NO: 50)

A2GL_HUMAN SPdb1223  Homo sapiens  (Human)

(SEQ ID NO: 51)

A2MG_HUMAN SPdb1225  Homo sapiens  (Human)

(SEQ ID NO: 52)

ABP1_HUMAN SPdb2047  Homo sapiens  (Human)

(SEQ ID NO: 53)

ACET_HUMAN SPdb2859  Homo sapiens  (Human)

(SEQ ID NO: 54)

ACHB_HUMAN SPdb2984  Homo sapiens  (Human)

(SEQ ID NO: 55)

ACHE_HUMAN SPdb2999  Homo sapiens  (Human)

(SEQ ID NO: 56)

ACRO_HUMAN SPdb4311  Homo sapiens  (Human)

(SEQ ID NO: 57)

ADA32_HUMAN SPdb5197  Homo sapiens  (Human)

(SEQ ID NO: 58)

ADIPO_HUMAN SPdb5938  Homo sapiens  (Human)

(SEQ ID NO: 59)

ADML_HUMAN SPdb5966  Homo sapiens  (Human)

(SEQ ID NO: 60)

AGAL_HUMAN SPdb6382  Homo sapiens  (Human)

(SEQ ID NO: 61)

AGR2_HUMAN SPdb6560  Homo sapiens  (Human)

(SEQ ID NO: 62)

AGR3_HUMAN SPdb6563  Homo sapiens  (Human)

(SEQ ID NO: 63)

AMBP_HUMAN SPdb8414  Homo sapiens  (Human)

(SEQ ID NO: 64)

AMTN_HUMAN SPdb9141  Homo sapiens  (Human)

(SEQ ID NO: 65)

ANF_HUMAN SPdb9494  Homo sapiens  (Human)

(SEQ ID NO: 66)

ANGI_HUMAN SPdb9521  Homo sapiens  (Human)

(SEQ ID NO: 67)

ANGL3_HUMAN SPdb9540  Homo sapiens  (Human)

(SEQ ID NO: 68)

ANGL7_HUMAN SPdb9551  Homo sapiens  (Human)

(SEQ ID NO: 69)

ANGT_HUMAN SPdb9580  Homo sapiens  (Human)

(SEQ ID NO: 70)

ANPRA_HUMAN SPdb9903  Homo sapiens  (Human)

(SEQ ID NO: 71)

APOA2_HUMAN SPdb10884  Homo sapiens  (Human)

(SEQ ID NO: 72)

APOA4_HUMAN SPdb10891  Homo sapiens  (Human)

(SEQ ID NO: 73)

APOA_HUMAN SPdb10900  Homo sapiens  (Human)

(SEQ ID NO: 74)

APOC1_HUMAN SPdb10907  Homo sapiens  (Human)

(SEQ ID NO: 75)

APOC2_HUMAN SPdb10917  Homo sapiens  (Human)

(SEQ ID NO: 76)

APOC3_HUMAN SPdb10924  Homo sapiens  (Human)

(SEQ ID NO: 77)

APOD_HUMAN SPdb10936  Homo sapiens  (Human)

(SEQ ID NO: 78)

APOE_HUMAN SPdb10946  Homo sapiens  (Human)

(SEQ ID NO: 79)

APOH_HUMAN SPdb10966  Homo sapiens  (Human)

(SEQ ID NO: 80)

APR3_HUMAN SPdb11011  Homo sapiens  (Human)

(SEQ ID NO: 81)

ASM_HUMAN SPdb16794  Homo sapiens  (Human)

(SEQ ID NO: 82)

ASPG_HUMAN SPdb17050 Homo sapiens (Human)

(SEQ ID NO: 83)

BAMBI_HUMAN SPdb23773  Homo sapiens  (Human)

(SEQ ID NO: 84)

BASI_HUMAN SPdb23850  Homo sapiens  (Human)

(SEQ ID NO: 85)

BGAL_HUMAN SPdb24876  Homo sapiens  (Human)

(SEQ ID NO: 86)

BGLR_HUMAN SPdb24971  Homo sapiens  (Human)

(SEQ ID NO: 87)

BOC_HUMAN SPdb25928  Homo sapiens  (Human)

(SEQ ID NO: 88)

BPIL1_HUMAN SPdb26090  Homo sapiens  (Human)

(SEQ ID NO: 89)

BPI_HUMAN SPdb26097  Homo sapiens  (Human)

(SEQ ID NO: 90)

BT3A3_HUMAN SPdb26592  Homo sapiens  (Human)

(SEQ ID NO: 91)

BTNL8_HUMAN SPdb26692  Homo sapiens  (Human)

(SEQ ID NO: 92)

C16L2_HUMAN SPdb27156  Homo sapiens  (Human)

(SEQ ID NO: 93)

C1QT5_HUMAN SPdb27240  Homo sapiens  (Human)

(SEQ ID NO: 94)

C1QT6_HUMAN SPdb27243  Homo sapiens  (Human)

(SEQ ID NO: 95)

C1R_HUMAN SPdb27257  Homo sapiens  (Human)

(SEQ ID NO: 96)

C1S_HUMAN SPdb27262  Homo sapiens  (Human)

(SEQ ID NO: 97)

C4BPA_HUMAN SPdb27346  Homo sapiens  (Human)

(SEQ ID NO: 98)

CA187_HUMAN SPdb27946  Homo sapiens  (Human)

(SEQ ID NO: 99)

CADM3_HUMAN SPdb28390  Homo sapiens  (Human)

(SEQ ID NO: 100)

CAH9_HUMAN SPdb28555  Homo sapiens  (Human)

(SEQ ID NO: 101)

CALCR_HUMAN SPdb28726  Homo sapiens  (Human)

(SEQ ID NO: 102)

CALRL_HUMAN SPdb28899  Homo sapiens  (Human)

(SEQ ID NO: 103)

CALR_HUMAN SPdb28916  Homo sapiens  (Human)

(SEQ ID NO: 104)

CAP7_HUMAN SPdb29157  Homo sapiens  (Human)

(SEQ ID NO: 105)

CART_HUMAN SPdb29961  Homo sapiens  (Human)

(SEQ ID NO: 106)

CASA1_HUMAN SPdb29986  Homo sapiens  (Human)

(SEQ ID NO: 107)

CASB_HUMAN SPdb30005  Homo sapiens  (Human)

(SEQ ID NO: 108)

CASK_HUMAN SPdb30048  Homo sapiens  (Human)

(SEQ ID NO: 109)

CATC_HUMAN SPdb30354  Homo sapiens  (Human)

(SEQ ID NO: 110)

CATE_HUMAN SPdb30381  Homo sapiens  (Human)

(SEQ ID NO: 111)

CATG_HUMAN SPdb30393  Homo sapiens  (Human)

(SEQ ID NO: 112)

CATW_HUMAN SPdb30490  Homo sapiens  (Human)

(SEQ ID NO: 113)

CBG_HUMAN SPdb30913  Homo sapiens  (Human)

(SEQ ID NO: 114)

CBLN3_HUMAN SPdb31375  Homo sapiens  (Human)

(SEQ ID NO: 115)

CBLN4_HUMAN SPdb31377  Homo sapiens  (Human)

(SEQ ID NO: 116)

CBPA1_HUMAN SPdb31413  Homo sapiens  (Human)

(SEQ ID NO: 117)

CBPA3_HUMAN SPdb31420  Homo sapiens  (Human)

(SEQ ID NO: 118)

CBPB1_HUMAN SPdb31459  Homo sapiens  (Human)

(SEQ ID NO: 119)

CBPN_HUMAN SPdb31531  Homo sapiens  (Human)

(SEQ ID NO: 120)

CCL11_HUMAN SPdb32635  Homo sapiens  (Human)

(SEQ ID NO: 121)

CCL15_HUMAN SPdb32644  Homo sapiens  (Human)

(SEQ ID NO: 122)

CCL19_HUMAN SPdb32652  Homo sapiens  (Human)

(SEQ ID NO: 123)

CCL1_HUMAN SPdb32654  Homo sapiens  (Human)

(SEQ ID NO: 124)

CCL22_HUMAN SPdb32663  Homo sapiens  (Human)

(SEQ ID NO: 125)

CCL24_HUMAN SPdb32668  Homo sapiens  (Human)

(SEQ ID NO: 126)

CCL2_HUMAN SPdb32684  Homo sapiens  (Human)

(SEQ ID NO: 127)

CCL4_HUMAN SPdb32700  Homo sapiens  (Human)

(SEQ ID NO: 128)

CCL5_HUMAN SPdb32709  Homo sapiens  (Human)

(SEQ ID NO: 129)

CCL7_HUMAN SPdb32717  Homo sapiens  (Human)

(SEQ ID NO: 130)

CD180_HUMAN SPdb33618  Homo sapiens  (Human)

(SEQ ID NO: 131)

CD1A_HUMAN SPdb33624  Homo sapiens  (Human)

(SEQ ID NO: 132)

CD244_HUMAN SPdb33672  Homo sapiens  (Human)

(SEQ ID NO: 133)

CD276_HUMAN SPdb33680  Homo sapiens  (Human)

(SEQ ID NO: 134)

CD27_HUMAN SPdb33684  Homo sapiens  (Human)

(SEQ ID NO: 135)

CD28_HUMAN SPdb33689  Homo sapiens  (Human)

(SEQ ID NO: 136)

CD2_HUMAN SPdb33718  Homo sapiens  (Human)

(SEQ ID NO: 137)

CD320_HUMAN SPdb33725  Homo sapiens  (Human)

(SEQ ID NO: 138)

CD34_HUMAN SPdb33729  Homo sapiens  (Human)

(SEQ ID NO: 139)

CD3E_HUMAN SPdb33763  Homo sapiens  (Human)

(SEQ ID NO: 140)

CD3G_HUMAN SPdb33770  Homo sapiens  (Human)

(SEQ ID NO: 141)

CD3Z_HUMAN SPdb33777  Homo sapiens  (Human)

(SEQ ID NO: 142)

CD45_HUMAN SPdb33804  Homo sapiens  (Human)

(SEQ ID NO: 143)

CD5L_HUMAN SPdb33861  Homo sapiens  (Human)

(SEQ ID NO: 144)

CD5_HUMAN SPdb33872  Homo sapiens  (Human)

(SEQ ID NO: 145)

CD83_HUMAN SPdb33912  Homo sapiens  (Human)

(SEQ ID NO: 146)

CD8B_HUMAN SPdb33924  Homo sapiens  (Human)

(SEQ ID NO: 147)

CD99_HUMAN SPdb33932  Homo sapiens  (Human)

(SEQ ID NO: 148)

CEAM1_HUMAN SPdb34729  Homo sapiens  (Human)

(SEQ ID NO: 149)

CER1_HUMAN SPdb35232  Homo sapiens  (Human)

(SEQ ID NO: 150)

CERU_HUMAN SPdb35241  Homo sapiens  (Human)

(SEQ ID NO: 151)

CETP_HUMAN SPdb35298  Homo sapiens  (Human)

(SEQ ID NO: 152)

CF126_HUMAN SPdb35387  Homo sapiens  (Human)

(SEQ ID NO: 153)

CFAB_HUMAN SPdb35492  Homo sapiens  (Human)

(SEQ ID NO: 154)

CFAH_HUMAN SPdb35506  Homo sapiens  (Human)

(SEQ ID NO: 155)

CFAI_HUMAN SPdb35508  Homo sapiens  (Human)

(SEQ ID NO: 156)

CH3L1_HUMAN SPdb36470  Homo sapiens  (Human)

(SEQ ID NO: 157)

CH3L2_HUMAN SPdb36476  Homo sapiens  (Human)

(SEQ ID NO: 158)

CHIT1_HUMAN SPdb37892  Homo sapiens  (Human)

(SEQ ID NO: 159)

CL3L1_HUMAN SPdb39759  Homo sapiens  (Human)

(SEQ ID NO: 160)

CLC11_HUMAN SPdb39795  Homo sapiens  (Human)

(SEQ ID NO: 161)

CLC14_HUMAN SPdb39798  Homo sapiens  (Human)

(SEQ ID NO: 162)

CLM1_HUMAN SPdb40173  Homo sapiens  (Human)

(SEQ ID NO: 163)

CLM9_HUMAN SPdb40190  Homo sapiens  (Human)

(SEQ ID NO: 164)

CLUS_HUMAN SPdb41741  Homo sapiens  (Human)

(SEQ ID NO: 165)

CMA1_HUMAN SPdb41778  Homo sapiens  (Human)

(SEQ ID NO: 166)

CMGA_HUMAN SPdb41825  Homo sapiens  (Human)

(SEQ ID NO: 167)

CO1A1_HUMAN SPdb42643  Homo sapiens  (Human)

(SEQ ID NO: 168)

CO2_HUMAN SPdb42671  Homo sapiens  (Human)

(SEQ ID NO: 169)

CO3_HUMAN SPdb42683  Homo sapiens  (Human)

(SEQ ID NO: 170)

CO4A2_HUMAN SPdb42702  Homo sapiens  (Human)

(SEQ ID NO: 171)

CO5A2_HUMAN SPdb42724  Homo sapiens  (Human)

(SEQ ID NO: 172)

CO6A1_HUMAN SPdb42733  Homo sapiens  (Human)

(SEQ ID NO: 173)

CO6_HUMAN SPdb42741  Homo sapiens  (Human)

(SEQ ID NO: 174)

CO7_HUMAN SPdb42748  Homo sapiens  (Human)

(SEQ ID NO: 175)

CO9A1_HUMAN SPdb42770  Homo sapiens  (Human)

(SEQ ID NO: 176)

CO9_HUMAN SPdb42781  Homo sapiens  (Human)

(SEQ ID NO: 177)

COGA1_HUMAN SPdb44971  Homo sapiens  (Human)

(SEQ ID NO: 178)

COLI_HUMAN SPdb45068  Homo sapiens  (Human)

(SEQ ID NO: 179)

COL_HUMAN SPdb45103  Homo sapiens  (Human)

(SEQ ID NO: 180)

CR2_HUMAN SPdb47985  Homo sapiens  (Human)

(SEQ ID NO: 181)

CRDL2_HUMAN SPdb48539  Homo sapiens  (Human)

(SEQ ID NO: 182)

CREG1_HUMAN SPdb48576  Homo sapiens  (Human)

(SEQ ID NO: 183)

CRHBP_HUMAN SPdb48678  Homo sapiens  (Human)

(SEQ ID NO: 184)

CRIM1_HUMAN SPdb48688  Homo sapiens  (Human)

(SEQ ID NO: 185)

CRIS1_HUMAN SPdb48712  Homo sapiens  (Human)

(SEQ ID NO: 186)

CRLF1_HUMAN SPdb48785  Homo sapiens  (Human)

(SEQ ID NO: 187)

CRP_HUMAN SPdb48875  Homo sapiens  (Human)

(SEQ ID NO: 188)

CSF2R_HUMAN SPdb49487  Homo sapiens  (Human)

(SEQ ID NO: 189)

CSF2_HUMAN SPdb49495  Homo sapiens  (Human)

(SEQ ID NO: 190)

CSF3R_HUMAN SPdb49500  Homo sapiens  (Human)

(SEQ ID NO: 191)

CSPG2_HUMAN SPdb50031  Homo sapiens  (Human)

(SEQ ID NO: 192)

CST9L_HUMAN SPdb50299  Homo sapiens  (Human)

(SEQ ID NO: 193)

CST9_HUMAN SPdb50302  Homo sapiens  (Human)

(SEQ ID NO: 194)

CTHR1_HUMAN SPdb50661  Homo sapiens  (Human)

(SEQ ID NO: 195)

CTRB1_HUMAN SPdb50822  Homo sapiens  (Human)

(SEQ ID NO: 196)

CXCL7_HUMAN SPdb52153  Homo sapiens  (Human)

(SEQ ID NO: 197)

CXL10_HUMAN SPdb52338  Homo sapiens  (Human)

(SEQ ID NO: 198)

CYTC_HUMAN SPdb56121  Homo sapiens  (Human)

(SEQ ID NO: 199)

CYTL1_HUMAN SPdb56131  Homo sapiens  (Human)

(SEQ ID NO: 200)

CYTN_HUMAN SPdb56136  Homo sapiens  (Human)

(SEQ ID NO: 201)

CYTS_HUMAN SPdb56151  Homo sapiens  (Human)

(SEQ ID NO: 202)

CYTT_HUMAN SPdb56153  Homo sapiens  (Human)

(SEQ ID NO: 203)

D103A_HUMAN SPdb56224  Homo sapiens  (Human)

(SEQ ID NO: 204)

DB127_HUMAN SPdb57844  Homo sapiens  (Human)

(SEQ ID NO: 205)

DCD_HUMAN SPdb58564  Homo sapiens  (Human)

(SEQ ID NO: 206)

DEF1_HUMAN SPdb60113  Homo sapiens  (Human)

(SEQ ID NO: 207)

DKK1_HUMAN SPdb62795  Homo sapiens  (Human)

(SEQ ID NO: 208)

DKK3_HUMAN SPdb62800  Homo sapiens  (Human)

(SEQ ID NO: 209)

DKK4_HUMAN SPdb62802  Homo sapiens  (Human)

(SEQ ID NO: 210)

DLK_HUMAN SPdb62960  Homo sapiens  (Human)

(SEQ ID NO: 211)

DLL4_HUMAN SPdb62971  Homo sapiens  (Human)

(SEQ ID NO: 212)

DNAS1_HUMAN SPdb64678  Homo sapiens  (Human)

(SEQ ID NO: 213)

ECP_HUMAN SPdb69940  Homo sapiens  (Human)

(SEQ ID NO: 214)

EDAR_HUMAN SPdb70101  Homo sapiens  (Human)

(SEQ ID NO: 215)

EFNB1_HUMAN SPdb71207  Homo sapiens  (Human)

(SEQ ID NO: 216)

EFNB3_HUMAN SPdb71214  Homo sapiens  (Human)

(SEQ ID NO: 217)

EGFL8_HUMAN SPdb72652  Homo sapiens  (Human)

(SEQ ID NO: 218)

EGFR_HUMAN SPdb72662  Homo sapiens  (Human)

(SEQ ID NO: 219)

EGLN_HUMAN SPdb72697  Homo sapiens  (Human)

(SEQ ID NO: 220)

ELA2A_HUMAN SPdb73024  Homo sapiens  (Human)

(SEQ ID NO: 221)

ELA2B_HUMAN SPdb73028  Homo sapiens  (Human)

(SEQ ID NO: 222)

ELAF_HUMAN SPdb73040  Homo sapiens  (Human)

(SEQ ID NO: 223)

ENPL_HUMAN SPdb75253  Homo sapiens  (Human)

(SEQ ID NO: 224)

ENPP7_HUMAN SPdb75282  Homo sapiens  (Human)

(SEQ ID NO: 225)

EPGN_HUMAN SPdb75639  Homo sapiens  (Human)

(SEQ ID NO: 226)

EPHB1_HUMAN SPdb75671  Homo sapiens  (Human)

(SEQ ID NO: 227)

EPHB6_HUMAN SPdb75686  Homo sapiens  (Human)

(SEQ ID NO: 228)

EPOR_HUMAN SPdb75734  Homo sapiens  (Human)

(SEQ ID NO: 229)

EPO_HUMAN SPdb75747  Homo sapiens  (Human)

(SEQ ID NO: 230)

ESAM_HUMAN SPdb76640  Homo sapiens  (Human)

(SEQ ID NO: 231)

EST1_HUMAN SPdb76818  Homo sapiens  (Human)

(SEQ ID NO: 232)

F13B_HUMAN SPdb78523  Homo sapiens  (Human)

(SEQ ID NO: 233)

FA111_HUMAN SPdb78712  Homo sapiens  (Human)

(SEQ ID NO: 234)

FA5_HUMAN SPdb78865  Homo sapiens  (Human)

(SEQ ID NO: 235)

FA8_HUMAN SPdb78981  Homo sapiens  (Human)

(SEQ ID NO: 236)

FCGR1_HUMAN SPdb81611  Homo sapiens  (Human)

(SEQ ID NO: 237)

FCGRN_HUMAN SPdb81622  Homo sapiens  (Human)

(SEQ ID NO: 238)

FCN1_HUMAN SPdb81643  Homo sapiens  (Human)

(SEQ ID NO: 239)

FCN3_HUMAN SPdb81652  Homo sapiens  (Human)

(SEQ ID NO: 240)

FCRL2_HUMAN SPdb81673  Homo sapiens  (Human)

(SEQ ID NO: 241)

FCRLA_HUMAN SPdb81677  Homo sapiens  (Human)

(SEQ ID NO: 242)

FETUA_HUMAN SPdb82655  Homo sapiens  (Human)

(SEQ ID NO: 243)

FGF19_HUMAN SPdb82742  Homo sapiens  (Human)

(SEQ ID NO: 244)

FGF21_HUMAN SPdb82761  Homo sapiens  (Human)

(SEQ ID NO: 245)

FGF23_HUMAN SPdb82765  Homo sapiens  (Human)

(SEQ ID NO: 246)

FGFR3_HUMAN SPdb82845  Homo sapiens  (Human)

(SEQ ID NO: 247)

FGFR4_HUMAN SPdb82851  Homo sapiens  (Human)

(SEQ ID NO: 248)

FGRL1_HUMAN SPdb82874  Homo sapiens  (Human)

(SEQ ID NO: 249)

FIBA_HUMAN SPdb83006  Homo sapiens  (Human)

(SEQ ID NO: 250)

FIBG_HUMAN SPdb83075  Homo sapiens  (Human)

(SEQ ID NO: 251)

FKB14_HUMAN SPdb83515  Homo sapiens  (Human)

(SEQ ID NO: 252)

FKBP2_HUMAN SPdb83570  Homo sapiens  (Human)

(SEQ ID NO: 253)

FLRT2_HUMAN SPdb84949  Homo sapiens  (Human)

(SEQ ID NO: 254)

FSHB_HUMAN SPdb87356  Homo sapiens  (Human)

(SEQ ID NO: 255)

FSTL1_HUMAN SPdb87403  Homo sapiens  (Human)

(SEQ ID NO: 256)

FSTL3_HUMAN SPdb87408  Homo sapiens  (Human)

(SEQ ID NO: 257)

FST_HUMAN SPdb87422  Homo sapiens  (Human)

(SEQ ID NO: 258)

FZD3_HUMAN SPdb88844  Homo sapiens  (Human)

(SEQ ID NO: 259)

G6B_HUMAN SPdb89267  Homo sapiens  (Human)

(SEQ ID NO: 260)

GALC_HUMAN SPdb90251  Homo sapiens  (Human)

(SEQ ID NO: 261)

GDN_HUMAN SPdb94028  Homo sapiens  (Human)

(SEQ ID NO: 262)

GELS_HUMAN SPdb94075  Homo sapiens  (Human)

(SEQ ID NO: 263)

GI24_HUMAN SPdb94473  Homo sapiens  (Human)

(SEQ ID NO: 264)

GLHA_HUMAN SPdb96435  Homo sapiens  (Human)

(SEQ ID NO: 265)

GLPA_HUMAN SPdb98215  Homo sapiens  (Human)

(SEQ ID NO: 266)

GLPB_HUMAN SPdb98238  Homo sapiens  (Human)

(SEQ ID NO: 267)

GLPE_HUMAN SPdb98310  Homo sapiens  (Human)

(SEQ ID NO: 268)

GLUC_HUMAN SPdb99110  Homo sapiens  (Human)

(SEQ ID NO: 269)

GNS_HUMAN SPdb100194  Homo sapiens  (Human)

(SEQ ID NO: 270)

GP1BA_HUMAN SPdb100593  Homo sapiens  (Human)

(SEQ ID NO: 271)

GPI8_HUMAN SPdb101405  Homo sapiens  (Human)

(SEQ ID NO: 272)

GPIX_HUMAN SPdb101411  Homo sapiens  (Human)

(SEQ ID NO: 273)

GPR56_HUMAN SPdb102058  Homo sapiens  (Human)

(SEQ ID NO: 274)

GPR97_HUMAN SPdb102106  Homo sapiens  (Human)

(SEQ ID NO: 275)

GRAB_HUMAN SPdb102370  Homo sapiens  (Human)

(SEQ ID NO: 276)

GREM1_HUMAN SPdb102718  Homo sapiens  (Human)

(SEQ ID NO: 277)

GROA_HUMAN SPdb102897  Homo sapiens  (Human)

(SEQ ID NO: 278)

GRP78_HUMAN SPdb102967  Homo sapiens  (Human)

(SEQ ID NO: 279)

GRP_HUMAN SPdb103369  Homo sapiens  (Human)

(SEQ ID NO: 280)

HA22_HUMAN SPdb106653  Homo sapiens  (Human)

(SEQ ID NO: 281)

HA23_HUMAN SPdb106655  Homo sapiens  (Human)

(SEQ ID NO: 282)

HA25_HUMAN SPdb106658  Homo sapiens  (Human)

(SEQ ID NO: 283)

HA27_HUMAN SPdb106660  Homo sapiens  (Human)

(SEQ ID NO: 284)

HB21_HUMAN SPdb106943  Homo sapiens  (Human)

(SEQ ID NO: 285)

HB23_HUMAN SPdb106948  Homo sapiens  (Human)

(SEQ ID NO: 286)

HB24_HUMAN SPdb106950  Homo sapiens  (Human)

(SEQ ID NO: 287)

HB25_HUMAN SPdb106952  Homo sapiens  (Human)

(SEQ ID NO: 288)

HB2B_HUMAN SPdb106955  Homo sapiens  (Human)

(SEQ ID NO: 289)

HB2C_HUMAN SPdb106957  Homo sapiens  (Human)

(SEQ ID NO: 290)

HB2K_HUMAN SPdb106966  Homo sapiens  (Human)

(SEQ ID NO: 291)

HEP2_HUMAN SPdb110024  Homo sapiens  (Human)

(SEQ ID NO: 292)

HGFA_HUMAN SPdb110611  Homo sapiens  (Human)

(SEQ ID NO: 293)

HIS1_HUMAN SPdb111008  Homo sapiens  (Human)

(SEQ ID NO: 294)

HIS3_HUMAN SPdb111571  Homo sapiens  (Human)

(SEQ ID NO: 295)

HLAE_HUMAN SPdb113899  Homo sapiens  (Human)

(SEQ ID NO: 296)

HPSE_HUMAN SPdb115516  Homo sapiens  (Human)

(SEQ ID NO: 297)

HPT_HUMAN SPdb115541  Homo sapiens  (Human)

(SEQ ID NO: 298)

HV103_HUMAN SPdb118845  Homo sapiens  (Human)

(SEQ ID NO: 299)

HV303_HUMAN SPdb118866  Homo sapiens  (Human)

(SEQ ID NO: 300)

I10R1_HUMAN SPdb119663  Homo sapiens  (Human)

(SEQ ID NO: 301)

I10R2_HUMAN SPdb119665  Homo sapiens  (Human)

(SEQ ID NO: 302)

I17RA_HUMAN SPdb119686  Homo sapiens  (Human)

(SEQ ID NO: 303)

I17RB_HUMAN SPdb119688  Homo sapiens  (Human)

(SEQ ID NO: 304)

I17RC_HUMAN SPdb119690  Homo sapiens  (Human)

(SEQ ID NO: 305)

I18BP_HUMAN SPdb119699  Homo sapiens  (Human)

(SEQ ID NO: 306)

I20RA_HUMAN SPdb119707  Homo sapiens  (Human)

(SEQ ID NO: 307)

I20RB_HUMAN SPdb119709  Homo sapiens  (Human)

(SEQ ID NO: 308)

I22RA_HUMAN SPdb119711  Homo sapiens  (Human)

(SEQ ID NO: 309)

IBP1_HUMAN SPdb119979  Homo sapiens  (Human)

(SEQ ID NO: 310)

IBP2_HUMAN SPdb119988  Homo sapiens  (Human)

(SEQ ID NO: 311)

IBP3_HUMAN SPdb119995  Homo sapiens  (Human)

(SEQ ID NO: 312)

IBP7_HUMAN SPdb120015  Homo sapiens  (Human)

(SEQ ID NO: 313)

IFN16_HUMAN SPdb122381  Homo sapiens  (Human)

(SEQ ID NO: 314)

IFNA2_HUMAN SPdb122393  Homo sapiens  (Human)

(SEQ ID NO: 315)

IFNA4_HUMAN SPdb122399  Homo sapiens  (Human)

(SEQ ID NO: 316)

IFNA5_HUMAN SPdb122401  Homo sapiens  (Human)

(SEQ ID NO: 317)

IFNA6_HUMAN SPdb122403  Homo sapiens  (Human)

(SEQ ID NO: 318)

IFNA7_HUMAN SPdb122405  Homo sapiens  (Human)

(SEQ ID NO: 319)

IFNK_HUMAN SPdb122481  Homo sapiens  (Human)

(SEQ ID NO: 320)

IFNW1_HUMAN SPdb122503  Homo sapiens  (Human)

(SEQ ID NO: 321)

IGF2_HUMAN SPdb122603  Homo sapiens  (Human)

(SEQ ID NO: 322)

IGFL1_HUMAN SPdb122611  Homo sapiens  (Human)

(SEQ ID NO: 323)

IGFL3_HUMAN SPdb122613  Homo sapiens  (Human)

(SEQ ID NO: 324)

IL10_HUMAN SPdb123103  Homo sapiens  (Human)

(SEQ ID NO: 325)

IL12A_HUMAN SPdb123133  Homo sapiens  (Human)

(SEQ ID NO: 326)

IL12B_HUMAN SPdb123153  Homo sapiens  (Human)

(SEQ ID NO: 327)

IL17F_HUMAN SPdb123200  Homo sapiens  (Human)

(SEQ ID NO: 328)

IL17_HUMAN SPdb123202  Homo sapiens  (Human)

(SEQ ID NO: 329)

IL19_HUMAN SPdb123221  Homo sapiens  (Human)

(SEQ ID NO: 330)

IL1R1_HUMAN SPdb123285  Homo sapiens  (Human)

(SEQ ID NO: 331)

IL1RA_HUMAN SPdb123295  Homo sapiens  (Human)

(SEQ ID NO: 332)

IL20_HUMAN SPdb123302  Homo sapiens  (Human)

(SEQ ID NO: 333)

IL21R_HUMAN SPdb123304  Homo sapiens  (Human)

(SEQ ID NO: 334)

IL22_HUMAN SPdb123314  Homo sapiens  (Human)

(SEQ ID NO: 335)

IL24_HUMAN SPdb123324  Homo sapiens  (Human)

(SEQ ID NO: 336)

G

IL25_HUMAN SPdb123327  Homo sapiens  (Human)

(SEQ ID NO: 337)

IL2RA_HUMAN SPdb123341  Homo sapiens  (Human)

(SEQ ID NO: 338)

IL2RB_HUMAN SPdb123348  Homo sapiens  (Human)

(SEQ ID NO: 339)

IL2RG_HUMAN SPdb123355  Homo sapiens  (Human)

(SEQ ID NO: 340)

IL2_HUMAN SPdb123376  Homo sapiens  (Human)

(SEQ ID NO: 341)

IL3_HUMAN SPdb123414  Homo sapiens  (Human)

(SEQ ID NO: 342)

IL4_HUMAN SPdb123438  Homo sapiens  (Human)

(SEQ ID NO: 343)

IL5RA_HUMAN SPdb123452  Homo sapiens  (Human)

(SEQ ID NO: 344)

IL6RA_HUMAN SPdb123469  Homo sapiens  (Human)

(SEQ ID NO: 345)

IL7RA_HUMAN SPdb123503  Homo sapiens  (Human)

(SEQ ID NO: 346)

IL7_HUMAN SPdb123506  Homo sapiens  (Human)

(SEQ ID NO: 347)

IL9_HUMAN SPdb123527  Homo sapiens  (Human)

(SEQ ID NO: 348)

ILRL1_HUMAN SPdb123591  Homo sapiens  (Human)

(SEQ ID NO: 349)

INAR2_HUMAN SPdb124806  Homo sapiens  (Human)

(SEQ ID NO: 350)

INHA_HUMAN SPdb124863  Homo sapiens  (Human)

(SEQ ID NO: 351)

INSL3_HUMAN SPdb125047  Homo sapiens  (Human)

(SEQ ID NO: 352)

INSL4_HUMAN SPdb125053  Homo sapiens  (Human)

(SEQ ID NO: 353)

INSL5_HUMAN SPdb125056  Homo sapiens  (Human)

(SEQ ID NO: 354)

INS_HUMAN SPdb125119  Homo sapiens  (Human)

(SEQ ID NO: 355)

IPSP_HUMAN SPdb125761  Homo sapiens  (Human)

(SEQ ID NO: 356)

IRBP_HUMAN SPdb126007  Homo sapiens  (Human)

(SEQ ID NO: 357)

ISK6_HUMAN SPdb126513  Homo sapiens  (Human)

(SEQ ID NO: 358)

ITA2B_HUMAN SPdb128319  Homo sapiens  (Human)

(SEQ ID NO: 359)

ITA2_HUMAN SPdb128324  Homo sapiens  (Human)

(SEQ ID NO: 360)

ITA3_HUMAN SPdb128328  Homo sapiens  (Human)

(SEQ ID NO: 361)

ITA4_HUMAN SPdb128331  Homo sapiens  (Human)

(SEQ ID NO: 362)

ITA6_HUMAN SPdb128340  Homo sapiens  (Human)

(SEQ ID NO: 363)

ITA7_HUMAN SPdb128342  Homo sapiens  (Human)

(SEQ ID NO: 364)

ITAE_HUMAN SPdb128352  Homo sapiens  (Human)

(SEQ ID NO: 365)

ITAL_HUMAN SPdb128355  Homo sapiens  (Human)

(SEQ ID NO: 366)

ITAV_HUMAN SPdb128362  Homo sapiens  (Human)

(SEQ ID NO: 367)

ITAX_HUMAN SPdb128364  Homo sapiens  (Human)

(SEQ ID NO: 368)

ITB2_HUMAN SPdb128379  Homo sapiens  (Human)

(SEQ ID NO: 369)

ITB4_HUMAN SPdb128387  Homo sapiens  (Human)

(SEQ ID NO: 370)

ITIH4_HUMAN SPdb128480  Homo sapiens  (Human)

(SEQ ID NO: 371)

JAM1_HUMAN SPdb128758  Homo sapiens  (Human)

(SEQ ID NO: 372)

JAM2_HUMAN SPdb128761  Homo sapiens  (Human)

(SEQ ID NO: 373)

JAM3_HUMAN SPdb128763  Homo sapiens  (Human)

(SEQ ID NO: 374)

JAML1_HUMAN SPdb128766  Homo sapiens  (Human)

(SEQ ID NO: 375)

KAZD1_HUMAN SPdb130734  Homo sapiens  (Human)

(SEQ ID NO: 376)

KI2L1_HUMAN SPdb132984  Homo sapiens  (Human)

(SEQ ID NO: 377)

KIRR2_HUMAN SPdb133175  Homo sapiens  (Human)

(SEQ ID NO: 378)

KLK3_HUMAN SPdb133637  Homo sapiens  (Human)

(SEQ ID NO: 379)

KLKB1_HUMAN SPdb133654  Homo sapiens  (Human)

(SEQ ID NO: 380)

KNG1_HUMAN SPdb133744  Homo sapiens  (Human)

(SEQ ID NO: 381)

KTEL1_HUMAN SPdb135048  Homo sapiens  (Human)

(SEQ ID NO: 382)

KV403_HUMAN SPdb135854  Homo sapiens  (Human)

(SEQ ID NO: 383)

KV404_HUMAN SPdb135855  Homo sapiens  (Human)

(SEQ ID NO: 384)

L1CAM_HUMAN SPdb135937  Homo sapiens  (Human)

(SEQ ID NO: 385)

LALBA_HUMAN SPdb136381  Homo sapiens  (Human)

(SEQ ID NO: 386)

LAMB1_HUMAN SPdb136411  Homo sapiens  (Human)

(SEQ ID NO: 387)

LAMC1_HUMAN SPdb136476  Homo sapiens  (Human)

(SEQ ID NO: 388)

LAMP1_HUMAN SPdb136491  Homo sapiens  (Human)

(SEQ ID NO: 389)

LAMP2_HUMAN SPdb136496  Homo sapiens  (Human)

(SEQ ID NO: 390)

LBP_HUMAN SPdb136757  Homo sapiens  (Human)

(SEQ ID NO: 391)

LCAT_HUMAN SPdb136799  Homo sapiens  (Human)

(SEQ ID NO: 392)

LCN1_HUMAN SPdb136934  Homo sapiens  (Human)

(SEQ ID NO: 393)

LCTL_HUMAN SPdb137082  Homo sapiens  (Human)

(SEQ ID NO: 394)

LEUK_HUMAN SPdb139543  Homo sapiens  (Human)

(SEQ ID NO: 395)

LG3BP_HUMAN SPdb140022  Homo sapiens  (Human)

(SEQ ID NO: 396)

LIF_HUMAN SPdb140701  Homo sapiens  (Human)

(SEQ ID NO: 397)

LIPG_HUMAN SPdb141547  Homo sapiens  (Human)

(SEQ ID NO: 398)

LIPP_HUMAN SPdb141584  Homo sapiens  (Human)

(SEQ ID NO: 399)

LIRA3_HUMAN SPdb141630  Homo sapiens  (Human)

(SEQ ID NO: 400)

LMAN1_HUMAN SPdb141882  Homo sapiens  (Human)

(SEQ ID NO: 401)

LPH_HUMAN SPdb142734  Homo sapiens  (Human)

(SEQ ID NO: 402)

LRC55_HUMAN SPdb144275  Homo sapiens  (Human)

(SEQ ID NO: 403)

LRRN1_HUMAN SPdb144478  Homo sapiens  (Human)

(SEQ ID NO: 404)

LSHB_HUMAN SPdb144551  Homo sapiens  (Human)

(SEQ ID NO: 405)

LUM_HUMAN SPdb145084  Homo sapiens  (Human)

(SEQ ID NO: 406)

LU_HUMAN SPdb145367  Homo sapiens  (Human)

(SEQ ID NO: 407)

LV605_HUMAN SPdb145410  Homo sapiens  (Human)

(SEQ ID NO: 408)

LY86_HUMAN SPdb145461  Homo sapiens  (Human)

(SEQ ID NO: 409)

LYAM2_HUMAN SPdb145485  Homo sapiens  (Human)

(SEQ ID NO: 410)

LYPA3_HUMAN SPdb145568  Homo sapiens  (Human)

(SEQ ID NO: 411)

LYPD6_HUMAN SPdb145583  Homo sapiens  (Human)

(SEQ ID NO: 412)

LYSC_HUMAN SPdb145736  Homo sapiens  (Human)

(SEQ ID NO: 413)

MBL2_HUMAN SPdb148469  Homo sapiens  (Human)

(SEQ ID NO: 414)

MCP_HUMAN SPdb149004  Homo sapiens  (Human)

(SEQ ID NO: 415)

MFAP4_HUMAN SPdb152514  Homo sapiens  (Human)

(SEQ ID NO: 416)

MGP_HUMAN SPdb152777  Homo sapiens  (Human)

(SEQ ID NO: 417)

MIA_HUMAN SPdb153344  Homo sapiens  (Human)

(SEQ ID NO: 418)

MIME_HUMAN SPdb153448  Homo sapiens  (Human)

(SEQ ID NO: 419)

MIP2A_HUMAN SPdb153896  Homo sapiens  (Human)

(SEQ ID NO: 420)

MIP2B_HUMAN SPdb153898  Homo sapiens  (Human)

(SEQ ID NO: 421)

MK_HUMAN SPdb154137  Homo sapiens  (Human)

(SEQ ID NO: 422)

MMP1_HUMAN SPdb154439  Homo sapiens  (Human)

(SEQ ID NO: 423)

MOTI_HUMAN SPdb155899  Homo sapiens  (Human)

(SEQ ID NO: 424)

MOX2R_HUMAN SPdb155983  Homo sapiens  (Human)

(SEQ ID NO: 425)

MPRD_HUMAN SPdb156360  Homo sapiens  (Human)

(SEQ ID NO: 426)

MPRI_HUMAN SPdb156380  Homo sapiens  (Human)

(SEQ ID NO: 427)

MPZL3_HUMAN SPdb156425  Homo sapiens  (Human)

(SEQ ID NO: 428)

MSMB_HUMAN SPdb158422  Homo sapiens  (Human)

(SEQ ID NO: 429)

MYP0_HUMAN SPdb164385  Homo sapiens  (Human)

(SEQ ID NO: 430)

NAGAB_HUMAN SPdb165267  Homo sapiens  (Human)

(SEQ ID NO: 431)

NELL2_HUMAN SPdb168581  Homo sapiens  (Human)

(SEQ ID NO: 432)

NET02_HUMAN SPdb168760  Homo sapiens  (Human)

(SEQ ID NO: 433)

NEU1_HUMAN SPdb168777  Homo sapiens  (Human)

(SEQ ID NO: 434)

NEU2_HUMAN SPdb168794  Homo sapiens  (Human)

(SEQ ID NO: 435)

NGAL_HUMAN SPdb169201  Homo sapiens  (Human)

(SEQ ID NO: 436)

NID1_HUMAN SPdb169544  Homo sapiens  (Human)

(SEQ ID NO: 437)

NID2_HUMAN SPdb169547  Homo sapiens  (Human)

(SEQ ID NO: 438)

NLGNX_HUMAN SPdb170305  Homo sapiens  (Human)

(SEQ ID NO: 439)

NMB_HUMAN SPdb170485  Homo sapiens  (Human)

(SEQ ID NO: 440)

NOV_HUMAN SPdb171434  Homo sapiens  (Human)

(SEQ ID NO: 441)

NPTN_HUMAN SPdb171815  Homo sapiens  (Human)

(SEQ ID NO: 442)

NPY_HUMAN SPdb171875  Homo sapiens  (Human)

(SEQ ID NO: 443)

NRP1_HUMAN SPdb172978  Homo sapiens  (Human)

(SEQ ID NO: 444)

NTRK2_HUMAN SPdb173863  Homo sapiens  (Human)

(SEQ ID NO: 445)

NXPH3_HUMAN SPdb177361  Homo sapiens  (Human)

(SEQ ID NO: 446)

OLFL1_HUMAN SPdb178511  Homo sapiens  (Human)

(SEQ ID NO: 447)

OTOR_HUMAN SPdb180733  Homo sapiens  (Human)

(SEQ ID NO: 448)

OXLA_HUMAN SPdb181241  Homo sapiens  (Human)

(SEQ ID NO: 449)

P3IP1_HUMAN SPdb181543  Homo sapiens  (Human)

(SEQ ID NO: 450)

PAHO_HUMAN SPdb182446  Homo sapiens  (Human)

(SEQ ID NO: 451)

PARM1_HUMAN SPdb184104  Homo sapiens  (Human)

(SEQ ID NO: 452)

PCDBA_HUMAN SPdb184878  Homo sapiens  (Human)

(SEQ ID NO: 453)

PCOC2_HUMAN SPdb185137  Homo sapiens  (Human)

(SEQ ID NO: 454)

PCYXL_HUMAN SPdb185474  Homo sapiens  (Human)

(SEQ ID NO: 455)

PDGFA_HUMAN SPdb185687  Homo sapiens  (Human)

(SEQ ID NO: 456)

PDIA1_HUMAN SPdb185732  Homo sapiens  (Human)

(SEQ ID NO: 457)

PDIA3_HUMAN SPdb185743  Homo sapiens  (Human)

(SEQ ID NO: 458)

PDYN_HUMAN SPdb186941  Homo sapiens  (Human)

(SEQ ID NO: 459)

PEBP4_HUMAN SPdb187068  Homo sapiens  (Human)

(SEQ ID NO: 460)

PECA1_HUMAN SPdb187074  Homo sapiens  (Human)

(SEQ ID NO: 461)

PG12B_HUMAN SPdb188887  Homo sapiens  (Human)

(SEQ ID NO: 462)

PGFRA_HUMAN SPdb189004  Homo sapiens  (Human)

(SEQ ID NO: 463)

PGFRB_HUMAN SPdb189010  Homo sapiens  (Human)

(SEQ ID NO: 464)

PGH1_HUMAN SPdb189021  Homo sapiens  (Human)

(SEQ ID NO: 465)

PGRP2_HUMAN SPdb189713  Homo sapiens  (Human)

(SEQ ID NO: 466)

PIGT_HUMAN SPdb191467  Homo sapiens  (Human)

(SEQ ID NO: 467)

PIP_HUMAN SPdb191771  Homo sapiens  (Human)

(SEQ ID NO: 468)

PLBL2_HUMAN SPdb192296  Homo sapiens  (Human)

(SEQ ID NO: 469)

PLF4_HUMAN SPdb192496  Homo sapiens  (Human)

(SEQ ID NO: 470)

PLOD1_HUMAN SPdb192587  Homo sapiens  (Human)

(SEQ ID NO: 471)

PORIM_HUMAN SPdb194575  Homo sapiens  (Human)

(SEQ ID NO: 472)

PPA6_HUMAN SPdb195079  Homo sapiens  (Human)

(SEQ ID NO: 473)

PPAP_HUMAN SPdb195174  Homo sapiens  (Human)

(SEQ ID NO: 474)

PPGB_HUMAN SPdb195573  Homo sapiens  (Human)

(SEQ ID NO: 475)

PPIB_HUMAN SPdb195662  Homo sapiens  (Human)

(SEQ ID NO: 476)

PRB4_HUMAN SPdb196833  Homo sapiens  (Human)

(SEQ ID NO: 477)

PRLR_HUMAN SPdb197321  Homo sapiens  (Human)

(SEQ ID NO: 478)

PRL_HUMAN SPdb197346  Homo sapiens  (Human)

(SEQ ID NO: 479)

PROK1_HUMAN SPdb198420  Homo sapiens  (Human)

(SEQ ID NO: 480)

PROK2_HUMAN SPdb198424  Homo sapiens  (Human)

(SEQ ID NO: 481)

PROP_HUMAN SPdb198449  Homo sapiens  (Human)

(SEQ ID NO: 482)

PROZ_HUMAN SPdb198539  Homo sapiens  (Human)

(SEQ ID NO: 483)

PRP1_HUMAN SPdb198570  Homo sapiens  (Human)

(SEQ ID NO: 484)

PRPC_HUMAN SPdb198723  Homo sapiens  (Human)

(SEQ ID NO: 485)

PRRT3_HUMAN SPdb198854  Homo sapiens  (Human)

(SEQ ID NO: 486)

PTGDS_HUMAN SPdb203865  Homo sapiens  (Human)

(SEQ ID NO: 487)

PTHY_HUMAN SPdb203993  Homo sapiens  (Human)

(SEQ ID NO: 488)

PTPRG_HUMAN SPdb204842  Homo sapiens  (Human)

(SEQ ID NO: 489)

PYY_HUMAN SPdb210515  Homo sapiens  (Human)

(SEQ ID NO: 490)

PZP_HUMAN SPdb210532  Homo sapiens  (Human)

(SEQ ID NO: 491)

REG1A_HUMAN SPdb217426  Homo sapiens  (Human)

(SEQ ID NO: 492)

REG3G_HUMAN SPdb217443  Homo sapiens  (Human)

(SEQ ID NO: 493)

RIB1_HUMAN SPdb220194  Homo sapiens  (Human)

(SEQ ID NO: 494)

RIB2_HUMAN SPdb220201  Homo sapiens  (Human)

(SEQ ID NO: 495)

RISC_HUMAN SPdb221907  Homo sapiens  (Human)

(SEQ ID NO: 496)

RNAS1_HUMAN SPdb241351  Homo sapiens  (Human)

(SEQ ID NO: 497)

RNAS4_HUMAN SPdb241408  Homo sapiens  (Human)

(SEQ ID NO: 498)

S39A6_HUMAN SPdb264754  Homo sapiens  (Human)

(SEQ ID NO: 499)

SAA4_HUMAN SPdb264973  Homo sapiens  (Human)

(SEQ ID NO: 500)

SAA_HUMAN SPdb264986  Homo sapiens  (Human)

(SEQ ID NO: 501)

SAMP_HUMAN SPdb265378  Homo sapiens  (Human)

(SEQ ID NO: 502)

SCG1_HUMAN SPdb266260  Homo sapiens  (Human)

(SEQ ID NO: 503)

SCRG1_HUMAN SPdb266759  Homo sapiens  (Human)

(SEQ ID NO: 504)

SEM4B_HUMAN SPdb269059  Homo sapiens  (Human)

(SEQ ID NO: 505)

SEM6B_HUMAN SPdb269079  Homo sapiens  (Human)

(SEQ ID NO: 506)

SEMG1_HUMAN SPdb269090  Homo sapiens  (Human)

(SEQ ID NO: 507)

SEMG2_HUMAN SPdb269095  Homo sapiens  (Human)

(SEQ ID NO: 508)

SEPP1_HUMAN SPdb269196  Homo sapiens  (Human)

(SEQ ID NO: 509)

SFRP2_HUMAN SPdb269743  Homo sapiens  (Human)

(SEQ ID NO: 510)

SFRP3_HUMAN SPdb269747  Homo sapiens  (Human)

(SEQ ID NO: 511)

SFTPG_HUMAN SPdb269980  Homo sapiens  (Human)

(SEQ ID NO: 512)

SG1D4_HUMAN SPdb270016  Homo sapiens  (Human)

(SEQ ID NO: 513)

SG3A1_HUMAN SPdb270025  Homo sapiens  (Human)

(SEQ ID NO: 514)

SHBG_HUMAN SPdb270301  Homo sapiens  (Human)

(SEQ ID NO: 515)

SIAL_HUMAN SPdb270534  Homo sapiens  (Human)

(SEQ ID NO: 516)

SIDT2_HUMAN SPdb270589  Homo sapiens  (Human)

(SEQ ID NO: 517)

SLAF6_HUMAN SPdb271065  Homo sapiens  (Human)

(SEQ ID NO: 518)

SLAF7_HUMAN SPdb271067  Homo sapiens  (Human)

(SEQ ID NO: 519)

SLAF8_HUMAN SPdb271069  Homo sapiens  (Human)

(SEQ ID NO: 520)

SLPI_HUMAN SPdb271309  Homo sapiens  (Human)

(SEQ ID NO: 521)

SMR3B_HUMAN SPdb271976  Homo sapiens  (Human)

(SEQ ID NO: 522)

SMS_HUMAN SPdb272029  Homo sapiens  (Human)

(SEQ ID NO: 523)

SODE_HUMAN SPdb272720  Homo sapiens  (Human)

(SEQ ID NO: 524)

SOSD1_HUMAN SPdb273214  Homo sapiens  (Human)

(SEQ ID NO: 525)

SOST_HUMAN SPdb273220  Homo sapiens  (Human)

(SEQ ID NO: 526)

SPIT1_HUMAN SPdb274564  Homo sapiens  (Human)

(SEQ ID NO: 527)

SPIT2_HUMAN SPdb274566  Homo sapiens  (Human)

(SEQ ID NO: 528)

SRCH_HUMAN SPdb275436  Homo sapiens  (Human)

(SEQ ID NO: 529)

SRGN_HUMAN SPdb275551  Homo sapiens  (Human)

(SEQ ID NO: 530)

STAT_HUMAN SPdb277447  Homo sapiens  (Human)

(SEQ ID NO: 531)

STC1_HUMAN SPdb277483  Homo sapiens  (Human)

(SEQ ID NO: 532)

TCO1_HUMAN SPdb290388  Homo sapiens  (Human)

(SEQ ID NO: 533)

TCO2_HUMAN SPdb290391  Homo sapiens  (Human)

(SEQ ID NO: 534)

TENA_HUMAN SPdb291090  Homo sapiens  (Human)

(SEQ ID NO: 535)

TETN_HUMAN SPdb291269  Homo sapiens  (Human)

(SEQ ID NO: 536)

TFF1_HUMAN SPdb291574  Homo sapiens  (Human)

(SEQ ID NO: 537)

TFF3_HUMAN SPdb291583  Homo sapiens  (Human)

(SEQ ID NO: 538)

TFPI1_HUMAN SPdb291601  Homo sapiens  (Human)

(SEQ ID NO: 539)

TGFR2_HUMAN SPdb291788  Homo sapiens  (Human)

(SEQ ID NO: 540)

THBG_HUMAN SPdb292195  Homo sapiens  (Human)

(SEQ ID NO: 541)

THYG_HUMAN SPdb293853  Homo sapiens  (Human)

(SEQ ID NO: 542)

TICN2_HUMAN SPdb293959  Homo sapiens  (Human)

(SEQ ID NO: 543)

TIE1_HUMAN SPdb293968  Homo sapiens  (Human)

(SEQ ID NO: 544)

TIE2_HUMAN SPdb293972  Homo sapiens  (Human)

(SEQ ID NO: 545)

TIMP1_HUMAN SPdb294965  Homo sapiens  (Human)

(SEQ ID NO: 546)

TIMP2_HUMAN SPdb294980  Homo sapiens  (Human)

(SEQ ID NO: 547)

TIMP3_HUMAN SPdb294987  Homo sapiens  (Human)

(SEQ ID NO: 548)

TINAL_HUMAN SPdb295015  Homo sapiens  (Human)

(SEQ ID NO: 549)

TLR1_HUMAN SPdb295471  Homo sapiens  (Human)

(SEQ ID NO: 550)

TLR3_HUMAN SPdb295485  Homo sapiens  (Human)

(SEQ ID NO: 551)

TLR4_HUMAN SPdb295493  Homo sapiens  (Human)

(SEQ ID NO: 552)

TLR5_HUMAN SPdb295500  Homo sapiens  (Human)

(SEQ ID NO: 553)

TM2D1_HUMAN SPdb295869  Homo sapiens  (Human)

(SEQ ID NO: 554)

TMIG2_HUMAN SPdb296142  Homo sapiens  (Human)

(SEQ ID NO: 555)

TMM25_HUMAN SPdb296188  Homo sapiens  (Human)

(SEQ ID NO: 556)

TMM46_HUMAN SPdb296232  Homo sapiens  (Human)

(SEQ ID NO: 557)

TMM66_HUMAN SPdb296282  Homo sapiens  (Human)

(SEQ ID NO: 558)

TMM9B_HUMAN SPdb296355  Homo sapiens  (Human)

(SEQ ID NO: 559)

TNFB_HUMAN SPdb296533  Homo sapiens  (Human)

(SEQ ID NO: 560)

TNR14_HUMAN SPdb296702  Homo sapiens  (Human)

(SEQ ID NO: 561)

TNR16_HUMAN SPdb296704  Homo sapiens  (Human)

(SEQ ID NO: 562)

TNR18_HUMAN SPdb296709  Homo sapiens  (Human)

(SEQ ID NO: 563)

TNR19_HUMAN SPdb296711  Homo sapiens  (Human)

(SEQ ID NO: 564)

TNR1B_HUMAN SPdb296718  Homo sapiens  (Human)

(SEQ ID NO: 565)

TNR5_HUMAN SPdb296744  Homo sapiens  (Human)

(SEQ ID NO: 566)

TNR6B_HUMAN SPdb296751  Homo sapiens  (Human)

(SEQ ID NO: 567)

TNR8_HUMAN SPdb296768  Homo sapiens  (Human)

(SEQ ID NO: 568)

TNR9_HUMAN SPdb296771  Homo sapiens  (Human)

(SEQ ID NO: 569)

TPO_HUMAN SPdb298326  Homo sapiens  (Human)

(SEQ ID NO: 570)

TPSNR_HUMAN SPdb298428  Homo sapiens  (Human)

(SEQ ID NO: 571)

TPSN_HUMAN SPdb298434  Homo sapiens  (Human)

(SEQ ID NO: 572)

TR10D_HUMAN SPdb298538  Homo sapiens  (Human)

(SEQ ID NO: 573)

TR11B_HUMAN SPdb298559  Homo sapiens  (Human)

(SEQ ID NO: 574)

TR19L_HUMAN SPdb298594  Homo sapiens  (Human)

(SEQ ID NO: 575)

TRBM_HUMAN SPdb298866  Homo sapiens  (Human)

(SEQ ID NO: 576)

TRFE_HUMAN SPdb299004  Homo sapiens  (Human)

(SEQ ID NO: 577)

TRFL_HUMAN SPdb299019  Homo sapiens  (Human)

(SEQ ID NO: 578)

TRY1_HUMAN SPdb303488  Homo sapiens  (Human)

(SEQ ID NO: 579)

TRY2_HUMAN SPdb303496  Homo sapiens  (Human)

(SEQ ID NO: 580)

TSHB_HUMAN SPdb303727  Homo sapiens  (Human)

(SEQ ID NO: 581)

TSP1_HUMAN SPdb303853  Homo sapiens  (Human)

(SEQ ID NO: 582)

TVA2_HUMAN SPdb304532  Homo sapiens  (Human)

(SEQ ID NO: 583)

TVB2_HUMAN SPdb304538  Homo sapiens  (Human)

(SEQ ID NO: 584)

TXD12_HUMAN SPdb304820  Homo sapiens  (Human)

(SEQ ID NO: 585)

TXND4_HUMAN SPdb304996  Homo sapiens  (Human)

(SEQ ID NO: 586)

TYRP1_HUMAN SPdb305383  Homo sapiens  (Human)

(SEQ ID NO: 587)

UROK_HUMAN SPdb311443  Homo sapiens  (Human)

(SEQ ID NO: 588)

UTER_HUMAN SPdb311731  Homo sapiens  (Human)

(SEQ ID NO: 589)

UTS2_HUMAN SPdb311840  Homo sapiens  (Human)

(SEQ ID NO: 590)

VCAM1_HUMAN SPdb314239  Homo sapiens  (Human)

(SEQ ID NO: 591)

VEGFA_HUMAN SPdb314841  Homo sapiens  (Human)

(SEQ ID NO: 592)

VEGFC_HUMAN SPdb314852  Homo sapiens  (Human)

(SEQ ID NO: 593)

VGFR3_HUMAN SPdb315610  Homo sapiens  (Human)

(SEQ ID NO: 594)

VMO1_HUMAN SPdb317004  Homo sapiens  (Human)

(SEQ ID NO: 595)

VSIG2_HUMAN SPdb318531  Homo sapiens  (Human)

(SEQ ID NO: 596)

VSIG4_HUMAN SPdb318533  Homo sapiens  (Human)

(SEQ ID NO: 597)

VSTM1_HUMAN SPdb318698  Homo sapiens  (Human)

(SEQ ID NO: 598)

VTNC_HUMAN SPdb318799  Homo sapiens  (Human)

(SEQ ID NO: 599)

VWF_HUMAN SPdb318878  Homo sapiens  (Human)

(SEQ ID NO: 600)

WISP2_HUMAN SPdb319573  Homo sapiens  (Human)

(SEQ ID NO: 601)

X3CL1_HUMAN SPdb320112  Homo sapiens  (Human)

(SEQ ID NO: 602)

XCL2_HUMAN SPdb320139  Homo sapiens  (Human)

(SEQ ID NO: 603)

YK001_HUMAN SPdb348747  Homo sapiens  (Human)

(SEQ ID NO: 604)

YQ001_HUMAN SPdb352137  Homo sapiens  (Human)

(SEQ ID NO: 605)

ZA2G_HUMAN SPdb354335  Homo sapiens  (Human)

(SEQ ID NO: 606)

ZG16_HUMAN SPdb354803  Homo sapiens  (Human)

(SEQ ID NO: 607)

ZP2_HUMAN SPdb355981  Homo sapiens  (Human)

(SEQ ID NO: 608)

A preferred expression system using each specific human secretory signal peptide listed above is a CHO cell expression system.

In some embodiments, the human secretory signal peptide can include any combination of the aforelisted secretory signal peptide. In some embodiments, the human secretory signal peptide can specifically exclude any one or more of the aforelisted secretory signal peptide. For the sake of providing a concise description of the invention, listing of the above human secretory signal peptides is not repeated. Nonetheless this invention encompasses specific exclusion of any one of the human secretory signal peptide or nucleic acid sequences encoding each one of the secretory signal peptide listed above.

For the sake of providing a concise description of the invention, listing of nucleic acid sequences encoding each one of the secretory signal peptide listed above is omitted. Nonetheless, such nucleic acid sequences are publicly available and can be readily ascertained by a person of ordinary skill in the art. One public source of such nucleic acid sequence information is, e.g., http<://>proline<dot>bic<dot>nus<dot>edu<dot>sg.

In some embodiments, for mammalian expression systems, a protrypsin leading sequence can also be used.

In some embodiments, for production of NELL1 and/or NELL2 peptides in mammalian cells (e.g., CHO cells), the expression system for NELL1 and/or NELL2 can include the nucleic acid or cDNA that expresses an endogenous NELL signal peptide. In some embodiments, the method and system described herein specifically excludes an endogenous NELL signal peptide.

Peptide Purification

In some embodiments, the invention includes a method purifying NELL1 and/or NELL2 peptides secreted into culture media, according to standard peptide purification protocols, including, but not limited to those described below.

The method can also include collecting secreted NELL peptides and/or purifying NELL peptides for use. Peptide products can be tested for activity in a variety of functional or expression assays. For example in any assay, if a NELL peptide has a significant effect over a control substance on a given parameter, the NELL peptides can be functional to effect the measured parameter in a functional or expression assay.

In one embodiment, whether a selected cell expresses a selected nucleic acid sequence to express and/or secrete a NELL peptide can be examined. In one embodiment, the presence, amount or and/or activity of NELL peptides can be examined.

In one embodiment, NELL peptides detected and quantified by any of a number of methods well known to those of skill in the art. These can include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.

In one embodiment, Western blot (immunoblot) analysis can be used to detect and quantify the presence of NELL peptide(s) in a selected sample. Western blot analysis is well known in the art.

The assays of this invention can be scored (as positive or negative or quantity of target polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring can depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by an enzymatic label. A clearly visible colored band or spot at the correct molecular weight can be scored as a positive result, while the absence of a clearly visible spot or band can be scored as a negative. The intensity of the band or spot can provide a quantitative measure of target polypeptide concentration.

NELL peptides, when in non-monomeric forms can be a large molecule and many of the techniques that work for purification of smaller proteins may not work as well for NELL peptides. The unusually large size of NELL oligomeric protein molecules causes it to be incompatible with the industry standard techniques used to purify smaller proteins, such as monoclonal antibodies and blood clotting factors. Specifically, the industry standard media is made by cross-linking agarose (e.g., agarose columns) into round beads with dead-end channels. The channels are lined with a functional group and interaction of this functional group with a protein forms the basis for purification. Because the channels are dead-ends, a protein molecule enters the channels by the forces of diffusion. As the protein of interest increases in size, diffusion into the channel slows and purification efficiently drops.

Instead of traditional porous media (e.g., agarose columns), other media not traditionally used for proteins may be used to purify NELL peptides in a more efficient fashion both in terms of speed and amount of NELL peptides purified/recovered. The present invention provides a method for purifying NELL peptides with a higher productivity. A low productivity in purification of protein is generally considered a production rate limiting factor in protein production. Therefore achieving a higher productivity is important in protein production.

In some embodiments, the method includes subjecting crude NELL peptides to membrane chromatography to obtain purified NELL peptides. In some embodiments, the method includes using commercially available purification media in membrane format. In some embodiments, the NELL peptide is NELL-1 peptide. In some embodiments, the NELL peptide is NELL-2 peptide. By employing membrane purification media, an efficient purification process for the NELL peptide was developed whereas analogous efforts with porous media was much less efficient.

The ion-exchange membrane can be anion-exchange membrane or cation-exchange membrane. It is not at all obvious that membrane chromatography can be used to purify NELL peptides.

Further, it is particularly surprising that cation-exchange chromatography is effective for NELL peptide purification. For example, analysis of the NELL-1 primary protein sequence indicates that its theoretical pI is 5.8. Under the pH ranges of 7-8.5 typically used to purify proteins, NELL-1 would be predicted to carry a net negative charge and therefore bind to anion exchange functional groups such as quaternary or secondary amines and not necessarily cation exchanges. Unexpectedly, however, NELL-1 is found to bind to both cation and anion exchange functional groups.

In some embodiments, purification of Nell-1 peptide/protein can be effected through a chromatography process using a medium including a metal cation bearing two, three or more charges, e.g., an earth metal cation, a rare earth metal cation. Examples of such metal ions are, e.g., Mg(++), Ca(++), Al(+++), and any rare earth ions Ln(+++). Such metal ions can be included in a buffer solution or eluting solution.

In some embodiments, purification can sequentially employ a cation exchange chromotography, an anion exchange chromotography and an hydroxyapatite medium. In other embodiments, it can also sequentially employ an anion exchange chromotography, a cation exchange chromotography and an hydroxyapatite medium. Alternatively, other combinations or steps can be introduced in between the aforementioned sequences.

The following examples are offered to illustrate, but not to limit the claimed invention.

In general, expression hosts can be bacteria, yeast and fungi, mammalian cells, plants, transgenic animals or it can also be cell-free expression systems such as those based on wheat germ or E. coli extracts. In general, expression elements can be Prokaryotic, Yeast, Mammalian and Plant promoters or viral promoters. Protein expression strategies can be: intra- or extracellular, fusion proteins and display strategies. Downstream processing of recombinant proteins can include: harvest, lysis, filtration, ultrafiltration, precipitation, and/or other protein processing/purification strategies that encompass protein capture, purification, polishing, and optimization.