Mutant cell lines and methods for producing enhanced levels of recombinant proteins

This application claims priority from U.S. Provisional Application No. 60/099,603, filed Sep. 9, 1998.

This invention was made, at least in part, with government support under United States Public Health Service Grants from the Department of Health and Human Services, National Cancer Institute (CA 44059) and the National Institute of Allergy and Infectious Diseases (AI 34039). The U.S. government has certain rights in the invention.

Protein therapeutic agents are among the most important human and animal health products to have emerged from the biotechnology revolution of the 1980's and 1990's. These protein therapeutic agents include interferons, growth factors such as colony stimulating factor and erythropoietin, and monoclonal antibodies.

Many protein therapeutics are produced by a process which involves introducing a nucleic acid which encodes the desired exogenous protein into a living cell, expressing the exogenous protein in the cell, and then recovering the protein, referred to hereinafter as the “recombinant protein” from the system. Although a number of different cell types, including bacteria, yeast, insect cells, and plant cells can be used in this process, there are several advantages to using mammalian cells.

The major advantage to using mammalian cells to produce recombinant proteins is that processing of the proteins is normal. In other words, the proteins which are expressed in mammalian cells typically are glycosylated, phosphorylated and have the signal peptides removed therefrom. As a result, the recombinant protein is more similar to the naturally-occurring protein and, thus, more likely to be biologically active and non-antigenic. In addition, the toxic products that are typically produced in bacteria, yeast, insect cells and plant cells are avoided. Moreover, purification of the recombinant protein is often easier since the processed protein is secreted into the culture medium by mammalian cells. Accordingly, agents such as interferon beta, which is used in treatment of multiple sclerosis, currently are produced in Chinese hamster ovary (CHO)cells.

Unfortunately, high levels of the exogenous protein are not normally produced when mammalian cells are used in the recombinant process. Accordingly, it is desirable to have new methods for enhancing the expression of exogenous proteins in mammalian cells. Immortalized mutant mammalian cell lines which are capable of producing greater amounts of recombinant, exogenous proteins than their corresponding non-mutant mammalian cell lines are also desirable.

The present invention provides immortalized mutant cell lines that are capable of producing enhanced levels of exogenous, recombinant proteins. The mutant cell lines comprise mammalian somatic cells having a homozygous disruption in the gene which encodes the endoribonuclease known as RNase L and a homygous disruption in the gene which encodes the double-stranded RNA dependent kinase known as PKR. As used herein a “disruption” is a deletion of all or a portion of the RNase L gene or the PKR gene, an addition of one or more nucleotides to the RNase L gene or the PKR gene, a substitution of one or more of the nucleotides in the RNase L gene or the PKR gene, or any combination thereof. Preferably, the disruptions are in a coding exon of the RNase L and PKR genes. As a result of the homozygous disruptions, the mutant cell line, referred to hereinafter as the “double knock-out” or “DKO” cell line lacks biologically active forms of both the RNase L enzyme and the PKR enzyme.

The present invention also provides methods for producing enhanced levels of recombinant proteins, or polypeptides, in mammalian cell systems. In one aspect the method employs cells of the DKO cell line and comprises transfecting the DKO cells with a nucleic acid, or polynucleotide, encoding a desired, exogenous protein, or polypeptide; expressing the exogenous protein in the cell; and isolating the exogenous protein from the system, i.e., from the transfected cells, the culture medium of the transfected cells, or both. Preferably, the DKO cells are also transfected with, i.e., co-transfected with, a nucleic acid encoding the PKR inhibitor, adenovirus VAI RNA.

In another aspect the method employs mutant cells having a homozyous disruption in the RNase L gene alone, referred to hereinafter as “RNase L null” cells. RNase L null cells lack functional RNase L enzyme. The RNase L null cells are transfected with a nucleic acid encoding a desired, exogenous protein. Then the exogenous protein is expressed in the cell and isolated from the system. Preferably, the RNase L cells are co-transfected with a nucleic acid encoding adenovirus VAI RNA, or a nucleic acid encoding a dominant negative PKR, or a combination thereof. The subsequent steps involve expressing the nucleic acids that have been transfected into the RNase L null cells and isolating or purifying the exogenous protein from the system.

In another aspect the methods employ mutant cells having a homozygous disruption in the PKR gene, hereinafter referred to as “PKR null” cells. PKR null cells lack a functional PKR enzyme. In one embodiment, the method comprises co-transfecting the PKR null cells with a nucleic acid encoding adenovirus VAI RNA and with a nucleic acid encoding a desired, exogenous protein, expressing the exogenous protein in the cell, and isolating the exogenous protein from the system. In another embodiment, the method comprises co-transfecting the PKR null cells with a nucleic acid encoding a dominant negative RNase L and with a nucleic acid encoding a desired, exogenous protein; expressing the dominant negative RNase L and the desired, exogenous protein in the cell; and isolating the exogenous protein from the system. Preferably, the PKR null cells are transfected with a nucleic acid encoding adenovirus VAI RNA and a nucleic acid encoding a dominant negative RNase L, as well as with the nucleic acid encoding the exogenous protein.

In another aspect, the method comprises cotransfecting mammalian cells with a nucleic acid encoding the desired exogenous protein and a nucleic acid encoding a dominant negative RNase L or a dominant negative PKR. Preferably, the mammalian cells are cotransfected with both a nucleic acid encoding a dominant negative PKR and a nucleic acid encoding a dominant negative RNase L. More preferably, the mammalian cells are also transfected with a nucleic acid encoding adenovirus VAI RNA. The subsequent steps involve expressing the nucleic acids that have been transfected into the RNase L null cells and isolating the exogenous protein from the system.

The present invention also relates to methods of making the double knock-out cell line.

FIG. 1 . Enhanced levels of RNAs are expressed from different genes transfected into RNase L −/− cells. Induction of (A) apobec, (B) β-galactosidase, (C) CD4-GC and (D) CD4-AT mRNAs in wild type (RNase L +/+ ; PKR +/+ ) and RNase L −/− cells. Total cellular RNA was isolated at 18 h after transient transfection with 2 μg of each cDNA separately; and with 30 μg loaded per lane. Differences in RNA loading were examined by comparing with 28S ribosomal RNA ethidium bromide staining (lower panels).

FIG. 2 . β-galactosidase is overexpressed RNase L(RNL) −/− and PKR −/− cells. Cells were transfected with 4 μg of pSV-β-galactosidase vector and the β-galactosidase assay was performed 18 h after transient transfection. β-galactosidase activity reported represents the relative absorbance units per 10 6 cells. Values are an average of 3 independent transfections and were derived from sets of duplicate transfections. Some error bars were too small to appear on the graph.

FIG. 3. A luciferase gene driven by a CMV promoter is overexpressed in RNase L −/− and PKR −/− cells. Cells were transfected as described in Materials and Methods and after 18 h luciferase activity was assayed. Luciferase activity reported represents the relative luminiscence units per 10 6 cells. Values were average of 6 independent transfections and were derived from averages of duplicate transfections. Some error bars were too small to appear on the graph.

FIG. 4. A luciferase gene driven by an SV40 promoter is overexpressed in RNase L −/− and PKR −/− cells. Cells were transfected as described in Materials and Methods and after 18 h luciferase activity was assayed. Luciferase activity reported represents the relative luminiscence units per 10 6 cells. Values are averages of 6 independent transfections and were derived from sets of duplicate transfection.

FIG. 5. A dominant negative lyS 296 to arg mutant PKR, PKRm, greatly stimulates expression of β-galactosidase in RNase L −/− cells. RNase L −/− cells were transiently co-transfected with 2 μg of pSV-β-galactosidase and different amount of pRcCMV/PKRm or pRcCMV. After 18 h β-galactosidase activity was measured. β-galactosidase activity represents values per 10 6 cells. Values are averages of an experiment performed in duplicate.

FIG. 6 . PKRm enhances expression of β-galactosidase in wild type and RNase L −/− cells but not in PKR −/− cells. Cell were transiently co-transfected with 4 μg of pSV-β-galactosidase and 4 μg of pRcCMV/PKRm or 4 μg of pRcCMV separately. After 18 h, β-galactosidase activity was measured. β-galactosidase represents values per 10 6 cells. Values are averages from 2 experiments each performed in duplicate. Data are reported as relative absorbance units.

FIG. 7 . Adenovirus VAI RNA greatly enhances β-galactosidase expression in RNase L −/− cells. RNase L −/− cells were transiently cotransfected with 2 μg of pSV-β-galactosidase and different amount of pAdVAntage (Promega Co.) encoding VAI RNA or pBR322 vector control. After 18 h, β-galactosidase activity was measured. β-galactosidase activity is per 10 6 cells. Values are averages from an experiment performed in duplicate.

FIG. 8 . VAI RNA enhances β-galactosidase expression in wild type, RNase L(RNL) −/− and in PKR −/− cells. Cell were transiently cotransfected with 4 μg of pSV-β-galactosidase and 4 μg of pAdVAntage or 4 μg of pBR322 separately. After 18 h, β-galactosidase activity was measured. Values are averages from 2 experiments performed in duplicate. Data are reported as relative absorbance units per 10 6 cells.

FIG. 9. A dominant negative mutant of murine RNase L, RNase L ZB1, enhances β-galactosidase expression in PKR −/− cells. PKR −/− cells were transiently co-transfected, as describe in Methods, with 2 μg of pSV-β-galactosidase and different amount of pCDNA/ZB1 or pCDNA. After 18 h, β-galactosidase activity was measured. Values are an average of an experiment performed in duplicate. Data reported as relative absorbance units per 10 6 cells.

FIG. 10 . Immortalized RNase L −/− ; PKR −/− cells overexpress β-galactosidase. The double knockout (DKO), RNase L −/− ; PKR −/− cells were transfected with 4 μg of pSV-β-galactosidase and β-galactosidase assay was performed 18 h after transient transfection. β-galactosidase activity was normalized against protein concentration. Results are reported as relative absorbance units per 10 6 cells. Values were averages of 5 independent transfections and were derived from sets of duplicate transfection. Some error bars were too small to appear above the graph.

FIG. 11 . VAI RNA enhances β-galactosidase expression in DKO cells. DKO cells were transiently cotransfected with 2 μg of pSV-β-galactosidase and different amounts of pAdVAntage or different amounts of pBR322. After 18 h, β-galactosidase activity was measured.

FIG. 12 is a graph showing that VAI RNA enhances β-galactosidase expression in wild type, RNase L(RNL) −/− , PKR −/− , and DKO cells. Cell were transiently cotransfected with 4 μg of pSV-β-galactosidase and 4 μg of pAdVAntage or 4 μg of pBR322 separately. After 18 h, β-galactosidase activity was measured.

FIG. 13 shows the nucleotide sequence, SEQ ID NO:1 of a cDNA encoding mouse RNase L, and the amino acid sequence, SEQ ID NO:2, of mouse RNase L.

In accordance with the present invention, methodologies and mammalian cell lines useful for the large scale production of recombinant proteins are provided. In one aspect, the methods employ mammalian cells in which the endoribonuclease RNase L has either been ablated or inhibited. RNase L, which is found in basal levels in most mammalian cells is an unusual endoribonuclease that requires allosteric effectors to catalyze the hydrolysis of single-stranded RNA. In another aspect the method employs cells in which both RNase L and the double-stranded RNA dependent kinase known as PKR have been ablated or inhibited. In another aspect, the methods employ mutant mammalian cells, preferably mutant mouse cells, lacking functional RNase L, functional PKR, or both and comprising or containing adenovirus VAI RNA. In accordance with the present invention, it has been discovered that methods which involve ablation or inhibition of both PKR and RNase L in mammalian cells greatly increases the expression of exogenous proteins in such cells as compared to mammalian cells having functional RNase L and functional PKR enzymes. Accordingly, the present methods are useful for producing enhanced levels of protein therapeutics in mammalian cells.

The Mutant Cell Line

In accordance with the present invention a novel mammalian cell line for producing high levels of a recombinant, exogenous protein has been discovered. The cell line, which preferably is an immortalized cell line, is made up of somatic cells having a homozygous disruption in the gene which encodes the endoribonuclease RNase L and a homozygous disruption in the gene which encodes the double-stranded RNA dependent kinase known as PKR. As used herein the “disruptions” are a deletion of all or a portion of the RNase L gene and the PKR gene, an addition of one or more nucleotides to the RNase L gene and the PKR gene, a substitution of one or more of the nucleotides in the RNase L gene and the PKR gene, or any combination thereof. Preferably, the disruptions are in a coding exon of the RNase L and PKR genes. As a result of the homozygous disruptions, the mutant cell line lacks functional RNase L and PKR enzymes.

The DKO cell line is obtained by isolating somatic cells from a mutant non-human mammal comprising a homozygous disruption in both the RNase L gene and the PKR gene thereof. For example fibroblasts can be obtained from such animals and immortalized using conventional methods. In one embodiment, the mutant cell line is a mouse fibroblast cell line Such cell line may be produced by intercrossing mice which are heterozygous for the RNase L gene with mice which are heterozygous for the PKR gene and isolating mouse embryo fibroblasts from embryos having the genotype RNase L −/− PKR −/− . Methods for intercrossing mice, isolating fibroblasts from the resulting embryos, and immortalizing such primary fibroblasts are well known in the art.

Methods for preparing mice having a heterozygous disruption in the PKR gene are described in Yang, Y. L. et al. “Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.” , EMBO J. 14, 6095-6106 (1995), which is specifically incorporated herein by reference. Methods for preparing mice having a heterozygous disruption in the RNase L gene or a homozygous disruption in the RNase L gene are described in Zhou, A. et al. “Interferon action and apoptosis are defective in mice devoid of 2′,5′-oligoadenylate-dependent RNase L.” EMBO J. 16, 6355-6363 (1997) which is specifically incorporated herein by reference. Such mutant mice are produced by introducing a transgene into their germline. As used herein, the “transgene” is a variant of the RNase L gene. The transgene comprises a deletion of all or a portion of the RNase L gene or, an addition of a heterologous nucleic acid sequence to the wild-type RNase L gene, or a substitution of one or more base pairs in the RNase L gene. Preferably, the heterologous nucleic acid sequence is a marker sequence. More preferably, the heterologous nucleic acid sequence comprises a marker sequence that is inserted in the reverse orientation as compared to the coding sequence of the RNase L gene. As used herein the term “marker sequence” refers to a nucleic acid sequence that is used (1) to disrupt expression of the RNase L gene and (2) to identify those cells that have incorporated the transgene into the genome thereof. Suitable marker sequences are those that encode an assayable or selectable product and include, for example, antibiotic resistance genes or genes that encode an assayable enzyme not typically found in the cell. Where the marker sequence encodes a protein, the marker sequence will also typically contain a promoter that regulates its expression. The methods for preparing such DNA constructs are well-known in the art and, typically, employ genomic libraries and standard recombinant techniques.

Preferably, the disruption is in coding exon 1, coding exon 2, coding exon 3 or coding exon 4 of an RNase L gene, more preferably coding exon 1 of the mouse RNase L gene. The nucleotide sequence of a cDNA which encodes mouse RNase L and the amino acid sequence of the mouse RNase L enzyme are shown in FIG. 13 . Coding exon 1 encodes amino acids 1 to 491; coding exon 2 encodes amino acids 492-519, and coding exon 3 encodes amino acids 520 through 611 of the wild-type mouse RNase L enzyme.

In one aspect the methods of the present invention comprise transfecting a mutant mammalian cell having a homozygous disruption in the RNase L gene thereof, or in the PKR gene thereof, or, preferably, in both the RNase L and PKR genes thereof with a nucleic acid having a sequence which encodes a desired exogenous protein, expressing the protein, and then isolating the exogenous protein that has been synthesized in the mutant cell, i.e., the recombinant protein. As used herein “endogenous” refers to proteins that are encoded by one of the genes that exist in the natural or unmodified genome of the mammal from which the mutant mammalian cell was derived. As used herein “exogenous” refers to proteins encoded by a nucleic acid which is introduced into the cell using recombinant technology procedures, i.e. the transfected nucleic acid. The amino acid sequence of the exogenous protein may identical to the amino acid sequence of an endogenous protein. Preferably, the amino acid sequence of the exogenous protein is different from the amino acid sequences of the endogenous proteins of the cell. Somatic cells having a homozygous disruption in both the RNase L gene and the PKR gene, i.e. DKO cells, are prepared as described above. Somatic cells having a homozygous disruption of the RNase L gene alone are prepared as described in Zhou et al. Somatic cells having a homozygous disruption of the PKR gene alone are prepared as described in Yang et al.

Preferably, the mutant cells also comprise adenovirus VAI RNA. Such mutant cells are made by transiently transfecting or stably transfecting the mutant cell with a nucleic acid having a sequence which encodes adenovirus VAI RNA. A vector suitable for transfecting the mutant cell with a nucleic acid encoding adenovirus VAI RNA is available from Promega and is sold under the product name pAdVAntage. The pAdVAntage vector also encodes VAII RNA of adenovirus.

In another aspect, the method comprises cotransfecting a mammalian cell with a nucleic acid encoding the desired protein, hereinafter referred to as the “first nucleic acid” and a nucleic acid having a sequence which encodes an inhibitor of PKR, referred to hereinafter as the “second nucleic acid”, or a nucleic acid encoding an inhibitor of RNase L, referred to hereinafter as the “third nucleic acid”, or with both the second nucleic acid and the third nucleic acid. Preferably, the cells are transfected with a nucleic acid encoding adenovirus VA 1 RNA. The cells are cultured under conditions suitable for expressing the desired, exogenous protein, the PKR inhibitor, or the RNase L inhibitor, and, if necessary, the adenovirus VA 1, RNA, and then the recombinant protein is isolated from the system.

Examples of second nucleic acids are nucleic acids which encode a dominant negative PKR and nucleic acids which encode adenovirus VA 1 RNA. As used herein a “dominant negative protein” is a mutant form of a protein which has the property of inhibiting the function of the endogenous, wild type form of the protein which corresponds to the mutant protein. Typically, dominant negative proteins have amino acid substitutions or are truncated forms of the wild type protein. One example of a dominant negative RNase L, ZB1, is described in Hassel, B. A., Zhou, A., Sotomayor, C., Maran, A. & Silverman, R. H., “A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon.”, EMBO J. 12, 3297-3304 (1993), which is specifically incorporated herein by reference. ZB1 is derived from murine RNase L, but lacks the 89 carboxy terminal amino acids. ZB1 protein binds the activator of RNase L, 2-5A, but lacks ribonuclease activity and is capable of inhibiting the function of wild type RNase L. An example of a dominant negative of PKR is PKR (Lys296 Arg). PKR (Lys296Arg) is described in Katze M G, Wambach M, Wong M L, Garfinkel M, Meurs E, Chong K, Williams B R, Hovanessian A G, Barber G N, “Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.”, Mol Cell Biol 11: 11,5497-505, November, 1991, which is specifically incorporated herein by reference. PKR (Lys296Arg) contains a single amino acid substitution in the protein kinase domain II which is responsible for binding the alpha and beta phosphoryl groups of ATP and is required for protein kinase activity.

Cells employed in such methods include mutant mammalian cells such as for example RNase null cells and PKR null cells, as well as wild-type mammalian cells such as for example, CHO cells. In those instances where RNase null cells are used, it is preferred that the cells be co transfected with a nucleic acid encoding the desired exogenous protein and with a nucleic acid encoding a dominant negative PKR. More preferably, such cells are also transfected with a nucleic acid encoding adenovirus VA I RNA. In those cases where PKR null cells are used, it is preferred that the cells be transfected with a nucleic acid encoding the desired exogenous protein and with a nucleic acid encoding a dominant negative RNase L. More preferably, such cells are also transfected with a nucleic acid encoding VAI RNA. In those cases where wild-type mammalian cells are used, the cells are co-transfected with a first nucleic acid encoding the desired endogenous protein and a second nucleic acid encoding a dominant negative mutant form of PKR or a dominant negative form of RNase L. Preferably, the cells are co-transfected with the first nucleic acid and one of the following combinations: (1) a nucleic acid encoding a dominant negative mutant form of PKR and a nucleic acid encoding VAI RNA, (2) a nucleic aid encoding a dominant negative mutant form of PKR and a nucleic acid encoding a dominant negative RNase L, or (3) a nucleic acid encoding a dominant negative RNase L and a nucleic acid encoding adenovirus VAI RNA. More preferably, the wild-type cells are co-transfected with the first nucleic acid, a nucleic acid encoding a dominant negative PKR, a nucleic acid encoding a dominant negative RNase L, and a nucleic acid encoding adenovirus VAI RNA.

Promoters for the Nucleic Acids

The first nucleic acid, second nucleic acid, and third nucleic acid are operatively linked to a promoter, preferably a constitutive promoter, which promotes expression of the nucleic acids. Useful promoters include the CMV promoter, the SV40 promoter, and the beta actin promoter. Preferably, the first nucleic acid, second nucleic acid and third nucleic acid are incorporated into a vector comprising the promoter, such as for example a plasmid. Plasmids comprising such promoters are available commercially. Vectors for transfection of the nucleic acids into the mammalian cells, preferably, also include transcription initiation, termination and enhancer sequences, as well as sequences that enhance translation efficiency, such as the Kozak sequence. Preferably, the vector also includes a marker gene for selecting for the presence of the vector in the cell.

Although the nucleic acids may be contained within the same vector, it is preferred that the first nucleic acid, the second nucleic acid, and the third nucleic acid each be on separate vectors. In such case, the cell may be transfected with the second nucleic acid, the third nucleic acid, or both prior to transfection with the first nucleic acid or concurrently with the first nucleic acid. Introduction of the vector or vectors into the mammalian cell can be accomplished using a variety of methods well known in the art, such as for example, retrovirus-mediated transduction, microinjection, calcium phosphate treatment, lipid-mediated transfection or electroporation.

Following introduction of the vector into the cell, the transfected cells are incubated under conditions which are suitable for expression of the exogenous protein. Thereafter, the recombinant protein, i.e., the exogenous protein, is isolated from the transfected cells and, if secreted, from the culture medium of the transfected cells using conventional procedures. Following expression of the exogenous protein and growth of the transfected host cell to an appropriate cell density, the cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification of the recombinant protein or polypeptide.

Conventional procedures for isolating recombinant proteins from transformed host cells, such as isolation by initial extraction from cell pellets or from cell culture medium, followed by salting-out, and one or more chromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, and high performance liquid chromatography (HPLC), and affinity chromatography may be used to isolate the recombinant protein.

The following non-limiting examples are presented to further illustrate the present invention.

The rates of endogenous protein synthesis in wild-type fibroblasts, PKR −/− cells, and RNase L −/− cells was measured by measuring the incorporation of radiolabeled methionine into the total protein of these cells during a one hour incubation period. Therates of endogenous protein synthesis in each of these cell types following transfection with a control vector or with a vector containing a sequence encoding the adenovirus VAI RNA was also determined. Specifically, 10×10 5 cells of each cell type were transfected with pAdVAntage vector or pBR322 (control vector) as described above. At 18 hours after transfection, the cells were incubated in medium containing 100 μCi/ml of 35 S-L methionine in serum free medium for 1 h at 37° C. Following removal of the media, the cell layers were rinsed four times with 2 ml cold 5%TCA to precipitate the proteins in the cells. The precipitated protein in each cell layer was then solubilized in 0.25 M NaOH at room temperature, neutralized, and the radioactivity determined in a liquid scintillation counter. The amount of radioactivity, obtained by averaging the results from three separate studies, incorporated into protein of the transfected and non-transfected cells is shown in Table 1 below.

As shown in Table 1, endogenous protein synthesis was not enhanced in PKR −/− cells or RNase L −/− cells as compared to wild-type cells. Moreover, endogenous protein synthesis was not enhanced in any of these three cell types by transfecting these cells with a nucleic acid encoding adenovirus VA I RNA and adenovirus VA II RNA.

The radiolabeled protein produced during a one hour incubation of cells from each cell type was also assayed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results, determined by visual observation, confirmed those shown in Table 1.

While the methods and mutant cells for producing high levels of recombinant protein have been described to some degree of particularity, various adaptations and modifications can be made without departing from the scope of the invention as defined in the appended claims.

1

attcggcacg aggaaggtgc caattactag ctcccttctt tattcgtgta ctgatgagat 60

gtcagaagac agaacataat cagcccaatc cctactccaa gactctcatt gtgtcccaaa 120

gaaacacacg tgtgcatttc ccaaggaaaa ggcattgagg acc atg gag acc ccg 175

Met Glu Thr Pro

1

gat tat aac aca cct cag ggt gga acc cca tca gcg gga agt cag agg 223

Asp Tyr Asn Thr Pro Gln Gly Gly Thr Pro Ser Ala Gly Ser Gln Arg

5 10 15 20

acc gtt gtc gaa gat gat tct tcg ttg atc aaa gct gtt cag aag gga 271

Thr Val Val Glu Asp Asp Ser Ser Leu Ile Lys Ala Val Gln Lys Gly

25 30 35

gat gtt gtc agg gtc cag caa ttg tta gaa aaa ggg gct gat gcc aat 319

Asp Val Val Arg Val Gln Gln Leu Leu Glu Lys Gly Ala Asp Ala Asn

40 45 50

gcc tgt gaa gac acc tgg ggc tgg aca cct ttg cac aac gca gtg caa 367

Ala Cys Glu Asp Thr Trp Gly Trp Thr Pro Leu His Asn Ala Val Gln

55 60 65

gct ggc agg gta gac att gtg aac ctc ctg ctt agt cat ggt gct gac 415

Ala Gly Arg Val Asp Ile Val Asn Leu Leu Leu Ser His Gly Ala Asp

70 75 80

cct cat cgg agg aag aag aat ggg gcc acc ccc ttc atc att gct ggg 463

Pro His Arg Arg Lys Lys Asn Gly Ala Thr Pro Phe Ile Ile Ala Gly

85 90 95 100

atc cag gga gat gtg aaa ctg ctc gag att ctc ctc tct tgt ggt gca 511

Ile Gln Gly Asp Val Lys Leu Leu Glu Ile Leu Leu Ser Cys Gly Ala

105 110 115

gac gtc aat gag tgt gac gag aac gga ttc acg gct ttc atg gaa gct 559

Asp Val Asn Glu Cys Asp Glu Asn Gly Phe Thr Ala Phe Met Glu Ala

120 125 130

gct gag cgt ggt aac gct gaa gcc tta aga ttc ctt ttt gct aag gga 607

Ala Glu Arg Gly Asn Ala Glu Ala Leu Arg Phe Leu Phe Ala Lys Gly

135 140 145

gcc aat gtg aat ttg cga cga cag aca acg aag gac aaa agg cga ttg 655

Ala Asn Val Asn Leu Arg Arg Gln Thr Thr Lys Asp Lys Arg Arg Leu

150 155 160

aag caa gga ggc gcc aca gct ctc atg agc gct gct gag aag ggc cac 703

Lys Gln Gly Gly Ala Thr Ala Leu Met Ser Ala Ala Glu Lys Gly His

165 170 175 180

ctg gaa gtc ctg aga att ctc ctc aat gac atg aag gca gaa gtc gat 751

Leu Glu Val Leu Arg Ile Leu Leu Asn Asp Met Lys Ala Glu Val Asp

185 190 195

gct cgg gac aac atg ggc aga aat gcc ctg atc cgt act ctg ctg aac 799

Ala Arg Asp Asn Met Gly Arg Asn Ala Leu Ile Arg Thr Leu Leu Asn

200 205 210

tgg gat tgt gaa aat gtg gag gag att act tca atc ctg att cag cac 847

Trp Asp Cys Glu Asn Val Glu Glu Ile Thr Ser Ile Leu Ile Gln His

215 220 225

ggg gct gat gtt aac gtg aga gaa gaa aga ggg aaa aca ccc ctc atc 895

Gly Ala Asp Val Asn Val Arg Glu Glu Arg Gly Lys Thr Pro Leu Ile

230 235 240

gca gca gtg gag agg aag cac aca ggc ttg gtg cag atg ctc ctg agt 943

Ala Ala Val Glu Arg Lys His Thr Gly Leu Val Gln Met Leu Leu Ser

245 250 255 260

cgg gaa ggc ata aac ata gat gcc agg gat aac gag ggc aag aca gct 991

Arg Glu Gly Ile Asn Ile Asp Ala Arg Asp Asn Glu Gly Lys Thr Ala

265 270 275

ctg cta att gct gtt gat aaa caa ctg aag gaa att gtc cag ttg ctt 1039

Leu Leu Ile Ala Val Asp Lys Gln Leu Lys Glu Ile Val Gln Leu Leu

280 285 290

ctt gaa aag gga gct gat aag tgt gac gat ctt gtt tgg ata gcc agg 1087

Leu Glu Lys Gly Ala Asp Lys Cys Asp Asp Leu Val Trp Ile Ala Arg

295 300 305

agg aat cat gac tat cac ctt gta aag ctt ctc ctc ctt tat gta gct 1135

Arg Asn His Asp Tyr His Leu Val Lys Leu Leu Leu Leu Tyr Val Ala

310 315 320

aat cct gac acc gac cct cct gct gga gac tgg tcg cct cac agt tca 1183

Asn Pro Asp Thr Asp Pro Pro Ala Gly Asp Trp Ser Pro His Ser Ser

325 330 335 340

cgt tgg ggg aca gcc ttg aaa agc ctc cac agt atg act cga ccc atg 1231

Arg Trp Gly Thr Ala Leu Lys Ser Leu His Ser Met Thr Arg Pro Met

345 350 355

att ggc aaa ctc aag atc ttc att cat gat gac tat aaa att gct ggc 1279

Ile Gly Lys Leu Lys Ile Phe Ile His Asp Asp Tyr Lys Ile Ala Gly

360 365 370

act tcc gaa ggg gct gtc tac cta ggg atc tat gac aat cga gaa gtg 1327

Thr Ser Glu Gly Ala Val Tyr Leu Gly Ile Tyr Asp Asn Arg Glu Val

375 380 385

gct gtg aag gtc ttc cgt gag aat agc cca cgt gga tgt aag gaa gtc 1375

Ala Val Lys Val Phe Arg Glu Asn Ser Pro Arg Gly Cys Lys Glu Val

390 395 400

tct tgt ctg cgg gac tgc ggt gac cac agt aac tta gtg gct ttc tat 1423

Ser Cys Leu Arg Asp Cys Gly Asp His Ser Asn Leu Val Ala Phe Tyr

405 410 415 420

gga aga gag gac gat aag ggc tgt tta tat gtg tgt gtg tcc ctg tgt 1471

Gly Arg Glu Asp Asp Lys Gly Cys Leu Tyr Val Cys Val Ser Leu Cys

425 430 435

gag tgg aca ctg gaa gag ttc ctg agg ttg ccc aga gag gaa cct gtg 1519

Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu Pro Arg Glu Glu Pro Val

440 445 450

gag aac ggg gaa gat aag ttt gcc cac agc atc cta tta tct ata ttt 1567

Glu Asn Gly Glu Asp Lys Phe Ala His Ser Ile Leu Leu Ser Ile Phe

455 460 465

gag ggt gtt caa aaa cta cac ttg cat gga tat tcc cat cag gac ctg 1615

Glu Gly Val Gln Lys Leu His Leu His Gly Tyr Ser His Gln Asp Leu

470 475 480

caa cca caa aac atc tta ata gat tcc aag aaa gct gtc cgg ctg gca 1663

Gln Pro Gln Asn Ile Leu Ile Asp Ser Lys Lys Ala Val Arg Leu Ala

485 490 495 500

gat ttt gat cag agc atc cga tgg atg gga gag tca cag atg gtc agg 1711

Asp Phe Asp Gln Ser Ile Arg Trp Met Gly Glu Ser Gln Met Val Arg

505 510 515

aga gac ttg gag gat ctt gga cgg ctg gtt ctc tac gtg gta atg aaa 1759

Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr Val Val Met Lys

520 525 530

ggt gag atc ccc ttt gag aca cta aag act cag aat gat gaa gtg ctg 1807

Gly Glu Ile Pro Phe Glu Thr Leu Lys Thr Gln Asn Asp Glu Val Leu

535 540 545

ctt aca atg tct cca gat gag gag act aag gac ctc att cat tgc ctg 1855

Leu Thr Met Ser Pro Asp Glu Glu Thr Lys Asp Leu Ile His Cys Leu

550 555 560

ttt tct cct gga gaa aat gtc aag aac tgc ctg gta gac ctg ctt ggc 1903

Phe Ser Pro Gly Glu Asn Val Lys Asn Cys Leu Val Asp Leu Leu Gly

565 570 575 580

cat cct ttc ttt tgg act tgg gag aac gcg tat aga aca ctc cgg aat 1951

His Pro Phe Phe Trp Thr Trp Glu Asn Ala Tyr Arg Thr Leu Arg Asn

585 590 595

gtg gga aat gaa tct gac atc aaa gta cgg aaa tgt aaa agt gat ctt 1999

Val Gly Asn Glu Ser Asp Ile Lys Val Arg Lys Cys Lys Ser Asp Leu

600 605 610

ctc aga cta ctg gac cat cag aca ctt gag cct ccc aga agc ttt gac 2047

Leu Arg Leu Leu Asp His Gln Thr Leu Glu Pro Pro Arg Ser Phe Asp

615 620 625

cag tgg aca tgt aag atc gac aaa aat gtt atg gat gaa atg aat cat 2095

Gln Trp Thr Cys Lys Ile Asp Lys Asn Val Met Asp Glu Met Asn His

630 635 640

ttc tac gaa aag aga aaa aaa aac cct tat cag gat act gta ggt gat 2143

Phe Tyr Glu Lys Arg Lys Lys Asn Pro Tyr Gln Asp Thr Val Gly Asp

645 650 655 660

ctg ctg aag ttt att cgg aat ata ggc gaa cac atc aat gag gaa aaa 2191

Leu Leu Lys Phe Ile Arg Asn Ile Gly Glu His Ile Asn Glu Glu Lys

665 670 675

aag cgg ggg 2200

Lys Arg Gly

2

679

RNase L

2

Met Glu Thr Pro Asp Tyr Asn Thr Pro Gln Gly Gly Thr Pro Ser Ala

1 5 10 15

Gly Ser Gln Arg Thr Val Val Glu Asp Asp Ser Ser Leu Ile Lys Ala

20 25 30

Val Gln Lys Gly Asp Val Val Arg Val Gln Gln Leu Leu Glu Lys Gly

35 40 45

Ala Asp Ala Asn Ala Cys Glu Asp Thr Trp Gly Trp Thr Pro Leu His

50 55 60

Asn Ala Val Gln Ala Gly Arg Val Asp Ile Val Asn Leu Leu Leu Ser

65 70 75 80

His Gly Ala Asp Pro His Arg Arg Lys Lys Asn Gly Ala Thr Pro Phe

85 90 95

Ile Ile Ala Gly Ile Gln Gly Asp Val Lys Leu Leu Glu Ile Leu Leu

100 105 110

Ser Cys Gly Ala Asp Val Asn Glu Cys Asp Glu Asn Gly Phe Thr Ala

115 120 125

Phe Met Glu Ala Ala Glu Arg Gly Asn Ala Glu Ala Leu Arg Phe Leu

130 135 140

Phe Ala Lys Gly Ala Asn Val Asn Leu Arg Arg Gln Thr Thr Lys Asp

145 150 155 160

Lys Arg Arg Leu Lys Gln Gly Gly Ala Thr Ala Leu Met Ser Ala Ala

165 170 175

Glu Lys Gly His Leu Glu Val Leu Arg Ile Leu Leu Asn Asp Met Lys

180 185 190

Ala Glu Val Asp Ala Arg Asp Asn Met Gly Arg Asn Ala Leu Ile Arg

195 200 205

Thr Leu Leu Asn Trp Asp Cys Glu Asn Val Glu Glu Ile Thr Ser Ile

210 215 220

Leu Ile Gln His Gly Ala Asp Val Asn Val Arg Glu Glu Arg Gly Lys

225 230 235 240

Thr Pro Leu Ile Ala Ala Val Glu Arg Lys His Thr Gly Leu Val Gln

245 250 255

Met Leu Leu Ser Arg Glu Gly Ile Asn Ile Asp Ala Arg Asp Asn Glu

260 265 270

Gly Lys Thr Ala Leu Leu Ile Ala Val Asp Lys Gln Leu Lys Glu Ile

275 280 285

Val Gln Leu Leu Leu Glu Lys Gly Ala Asp Lys Cys Asp Asp Leu Val

290 295 300

Trp Ile Ala Arg Arg Asn His Asp Tyr His Leu Val Lys Leu Leu Leu

305 310 315 320

Leu Tyr Val Ala Asn Pro Asp Thr Asp Pro Pro Ala Gly Asp Trp Ser

325 330 335

Pro His Ser Ser Arg Trp Gly Thr Ala Leu Lys Ser Leu His Ser Met

340 345 350

Thr Arg Pro Met Ile Gly Lys Leu Lys Ile Phe Ile His Asp Asp Tyr

355 360 365

Lys Ile Ala Gly Thr Ser Glu Gly Ala Val Tyr Leu Gly Ile Tyr Asp

370 375 380

Asn Arg Glu Val Ala Val Lys Val Phe Arg Glu Asn Ser Pro Arg Gly

385 390 395 400

Cys Lys Glu Val Ser Cys Leu Arg Asp Cys Gly Asp His Ser Asn Leu

405 410 415

Val Ala Phe Tyr Gly Arg Glu Asp Asp Lys Gly Cys Leu Tyr Val Cys

420 425 430

Val Ser Leu Cys Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu Pro Arg

435 440 445

Glu Glu Pro Val Glu Asn Gly Glu Asp Lys Phe Ala His Ser Ile Leu

450 455 460

Leu Ser Ile Phe Glu Gly Val Gln Lys Leu His Leu His Gly Tyr Ser

465 470 475 480

His Gln Asp Leu Gln Pro Gln Asn Ile Leu Ile Asp Ser Lys Lys Ala

485 490 495

Val Arg Leu Ala Asp Phe Asp Gln Ser Ile Arg Trp Met Gly Glu Ser

500 505 510

Gln Met Val Arg Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr

515 520 525

Val Val Met Lys Gly Glu Ile Pro Phe Glu Thr Leu Lys Thr Gln Asn

530 535 540

Asp Glu Val Leu Leu Thr Met Ser Pro Asp Glu Glu Thr Lys Asp Leu

545 550 555 560

Ile His Cys Leu Phe Ser Pro Gly Glu Asn Val Lys Asn Cys Leu Val

565 570 575

Asp Leu Leu Gly His Pro Phe Phe Trp Thr Trp Glu Asn Ala Tyr Arg

580 585 590

Thr Leu Arg Asn Val Gly Asn Glu Ser Asp Ile Lys Val Arg Lys Cys

595 600 605

Lys Ser Asp Leu Leu Arg Leu Leu Asp His Gln Thr Leu Glu Pro Pro

610 615 620

Arg Ser Phe Asp Gln Trp Thr Cys Lys Ile Asp Lys Asn Val Met Asp

625 630 635 640

Glu Met Asn His Phe Tyr Glu Lys Arg Lys Lys Asn Pro Tyr Gln Asp

645 650 655

Thr Val Gly Asp Leu Leu Lys Phe Ile Arg Asn Ile Gly Glu His Ile

660 665 670

Asn Glu Glu Lys Lys Arg Gly

675

3

28

RNase L

3

gtttggctat ttctctgtgt tcattgga 28

4

26

RNase L

4

gtaatggcta ctccgtgcat ctgggc 26

5

27

RNase L

5

attcgcagcg catcgccttc tatcgcc 27