A method for identifying at least a first unknown active domain in a region of known amino acid sequence of a parent polypeptide, which parent polypeptide has been cloned and has a pre-identified biological activity, said active domain capable of interacting with a first target when said parent polypeptide is in its native-folded form, which interaction is responsible for said biological activity, said method comprising: (a) comparing amino acid sequence or polypeptide structure in the region of known amino acid sequence of the parent polypeptide with amino acid sequence or polypeptide structure in a region of known amino acid sequence of an analog polypeptide to said parent polypeptide, said analog having at least 15% sequence homology with said parent polypeptide or .alpha.-carbon coordinates within about 2 to about 3.5 .ANG. of parent polypeptide .alpha.-carbon coordinates for about 60% or more of the analog sequence, wherein any interaction of said analog with said first target is different from the interaction of said parent polypeptide with said first target; (b) substituting DNA encoding a first analogous polypeptide segment from said analog into DNA encoding substantially the full-length parent polypeptide and expressing a first segment-substituted polypeptide; (c) contacting said first segment-substituted polypeptide with said first target to determine the interaction, if any, between said first target and said segment-substituted polypeptide; (d) repeating steps (b) and (c) using a second analogous polypeptide segment from an analog to said parent polypeptide to form at least a second segment-substituted polypeptide containing said second analogous polypeptide segment, which is different from said first analogous polypeptide segment; and (e) comparing the difference, if any, between the activity relative to said first target of said parent polypeptide and said first and second segment-substituted polypeptides as an indication of the location of said first unknown active domain in said parent polypeptide.
›2.↳ 1The method of claim 1 wherein said first unknown active domain comprises at least two discontinuous amino acid segments in the primary amino acid sequ…d2
The method of claim 1 wherein said first unknown active domain comprises at least two discontinuous amino acid segments in the primary amino acid sequence of said parent polypeptide.
›3.↳ 1The method of claim 1 wherein at least a first selected polypeptide segment of said parent polypeptide replaced by said first analogous polypeptide se…d2+1
The method of claim 1 wherein at least a first selected polypeptide segment of said parent polypeptide replaced by said first analogous polypeptide segment of said analog contains at least one amino acid residue located on the surface of the native-folded form of said parent polypeptide.
›4.↳ 3The method of claim 3 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, conta…d3
The method of claim 3 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of substantially all of the amino acid residues on said surface of said parent polypeptide.
›5.↳ 1The method of claim 1 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, conta…d2
The method of claim 1 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of analogous polypeptide segments covering about 15-100% of the amino acid sequence of said parent polypeptide.
›6.↳ 1The method of claim 1 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, conta…d2
The method of claim 1 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of analogous polypeptide segments covering about 60-100% of the amino acid sequence of said parent polypeptide.
›7.↳ 1The method of claim 1 further comprising identifying a second unknown active domain of said parent polypeptide, said second active domain interacting …d2
The method of claim 1 further comprising identifying a second unknown active domain of said parent polypeptide, said second active domain interacting with a second target, said method comprising repeating steps (b) through (e) with said second target.
›8.↳ 1The method of claim 1 further comprising identifying at least a first active amino acid residue within said first active domain, said method comprisin…d2+9
The method of claim 1 further comprising identifying at least a first active amino acid residue within said first active domain, said method comprising: (f) substituting a scanning amino acid for a different first amino acid residue within said first active domain to form a first residue-substituted polypeptide; (g) contacting said first residue-substituted polypeptide with said first target to determine the interaction, if any, between said first target and said first residue-substituted polypeptide; (h) repeating steps (f) and (g) to substitute a scanning amino acid for at least a second amino acid residue within said first active domain to form at least a second residue-substituted polypeptide; and (i) comparing the difference, if any, between the activity relative to said first target of said parent polypeptide and each of said first and second residue-substituted polypeptides as an indication of the location of said first active amino acid residue in said first active domain.
›9.↳ 8The method of claim 8 further comprising repeating steps (b) through (i) with a second target to identity a second active domain and at least one acti…d3+4
The method of claim 8 further comprising repeating steps (b) through (i) with a second target to identity a second active domain and at least one active amino acid residue within said second active domain.
›10.↳ 9The method of claim 9 further comprising the step of substituting an active amino acid residue in said first active domain with a different amino acid…d4+1
The method of claim 9 further comprising the step of substituting an active amino acid residue in said first active domain with a different amino acid to produce a polypeptide variant having a modified interaction with said first target but which retains substantially all of the interaction of said parent polypeptide with said second target.
›11.↳ 10The method of claim 10 further comprising the step of substituting an active amino acid residue in said second active domain with a different amino ac…d5
The method of claim 10 further comprising the step of substituting an active amino acid residue in said second active domain with a different amino acid to produce a polypeptide variant having a modified interaction with said second target but which retains the interaction of said parent polypeptide with said first target.
›12.↳ 9The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further compri…d4
The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further comprising substituting at least said one common active amino acid residue with a different amino acid to produce a polypeptide variant having modified interactions with each of said first and said second targets.
›13.↳ 9The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further compri…d4
The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further comprising substituting at least one amino acid residue in said first active domain, other than said at least one common active amino acid residue, with a different amino acid to produce a polypeptide variant having a modified interaction with said first target.
›44.↳ 8The method of claim 8 wherein at least one of said scanning amino acids is an isosteric amino acid.d3
The method of claim 8 wherein at least one of said scanning amino acids is an isosteric amino acid.
›45.↳ 8The method of claim 8 wherein the same scanning amino acid is employed in steps (f) and (h), and said scanning amino acid is a neutral amino acid.d3+2
The method of claim 8 wherein the same scanning amino acid is employed in steps (f) and (h), and said scanning amino acid is a neutral amino acid.
›46.↳ 45The method of claim 45 wherein said neutral amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.d4+1
The method of claim 45 wherein said neutral amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.
›47.↳ 46The method of claim 46 wherein said scanning amino acid is alanine.d5
The method of claim 46 wherein said scanning amino acid is alanine.
›27.↳ 1The method of claim 1 wherein said parent polypeptide is selected from the group consisting of growth hormone, prolactin, placental lactogen, .alpha.-…d2+1
The method of claim 1 wherein said parent polypeptide is selected from the group consisting of growth hormone, prolactin, placental lactogen, .alpha.-interferon, .gamma.-interferon, TGF-.alpha..sub.1, EGF, IGF-1, GM-CSF, TNF, tissue plasminogen activator, and CD-4 receptor.
›28.↳ 27The method of claim 27 wherein said parent polypeptide is selected from the group consisting of human growth hormone, human placental lactogen, and hu…d3
The method of claim 27 wherein said parent polypeptide is selected from the group consisting of human growth hormone, human placental lactogen, and human prolactin.
›29.↳ 1The method of claim 1 wherein said activity is measured in an in vitro assay.d2+3
The method of claim 1 wherein said activity is measured in an in vitro assay.
›30.↳ 29The method of claim 29 wherein said parent polypeptide is a hormone and said activity is measured in an in vitro assay using a soluble hormone recepto…d3+2
The method of claim 29 wherein said parent polypeptide is a hormone and said activity is measured in an in vitro assay using a soluble hormone receptor.
›31.↳ 30The method of claim 30 wherein said hormone is human growth hormone and said soluble hormone receptor is shGHr.d4
The method of claim 30 wherein said hormone is human growth hormone and said soluble hormone receptor is shGHr.
›32.↳ 30The method of claim 30 wherein said hormone is human growth hormone and said soluble hormone receptor is shPRLr.d4
The method of claim 30 wherein said hormone is human growth hormone and said soluble hormone receptor is shPRLr.
›33.↳ 1The method of claim 1 wherein said interaction between said first target and said parent polypeptide involves either binding or catalytic interaction …d2+2
The method of claim 1 wherein said interaction between said first target and said parent polypeptide involves either binding or catalytic interaction of said parent polypeptide with said first target.
›34.↳ 33The method of claim 33 wherein the activity between said first target and any of said substituted polypeptides is increased greater than two-fold as c…d3
The method of claim 33 wherein the activity between said first target and any of said substituted polypeptides is increased greater than two-fold as compared to said parent polypeptide.
›35.↳ 33The method of claim 33 wherein the activity between said first target and any of said substituted polypeptides is decreased greater than two-fold as c…d3
The method of claim 33 wherein the activity between said first target and any of said substituted polypeptides is decreased greater than two-fold as compared to said parent polypeptide.
›36.↳ 1The method of claim 1, wherein said analog has at least 15% amino acid sequence homology with said parent polypeptide.d2
The method of claim 1, wherein said analog has at least 15% amino acid sequence homology with said parent polypeptide.
›37.↳ 1The method of claim 1, wherein said analog is naturally occurring.d2
The method of claim 1, wherein said analog is naturally occurring.
›38.↳ 1The method of claim 1, wherein said analog is a tertiary analog.d2
The method of claim 1, wherein said analog is a tertiary analog.
›39.↳ 1The method of claim 1 wherein said parent polypeptide is human growth hormone and said analog is selected from the group consisting of human placental…d2
The method of claim 1 wherein said parent polypeptide is human growth hormone and said analog is selected from the group consisting of human placental lactogen, porcine growth hormone, and human prolactin.
›40.↳ 1The method of claim 1 wherein the biological activity of the parent polypeptide is of clinical utility.d2
The method of claim 1 wherein the biological activity of the parent polypeptide is of clinical utility.
›41.↳ 1The method of claim 1 wherein said parent polypeptide is selected from the group consisting of a hormone, enzyme, antigen, receptor, enzyme substrate,…d2
The method of claim 1 wherein said parent polypeptide is selected from the group consisting of a hormone, enzyme, antigen, receptor, enzyme substrate, binding protein, and enzyme inhibitor.
›42.↳ 1The method of claim 1 wherein said first target is selected from the group consisting of a hormone, enzyme, antibody, antigen, receptor, enzyme substr…d2
The method of claim 1 wherein said first target is selected from the group consisting of a hormone, enzyme, antibody, antigen, receptor, enzyme substrate, binding protein, and enzyme inhibitor.
›43.↳ 1The method of claim 1 wherein said analog does not substantially interact with said first target.d2
The method of claim 1 wherein said analog does not substantially interact with said first target.
›48.↳ 1The method of claim 1 wherein said parent polypeptide is naturally occurring.d2
The method of claim 1 wherein said parent polypeptide is naturally occurring.
