Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains

Abstract

Novel polypeptides having the scaffold structure of a C-type lectin-like domain (CTLD) and a randomized loop region for specifically binding a variety of target compounds and also provides nucleic acids encoding the polypeptides. Combinatorial CTLD libraries, methods for constructing the libraries, and methods for screening the libraries to identify and isolate the novel CTLD polypeptides. Libraries of nucleic acids encoding polypeptides having a scaffold CTLD with a randomized loop region, as well as nucleic acid sequences, vectors, and methods for preparing and expressing the libraries. Exemplary nucleic acids useful in the combinatorial libraries are derived from tetranectin and other proteins having a CTLD.

1 - 76 . (canceled) 
     
     
         77 . A combinatorial library comprising an ensemble of variant C-type lectin-like domain (CTLD) polypeptides having the scaffold structure of a CTLD polypeptide and a randomized CTLD loop region, the CTLD scaffold comprising the following structural elements:
 five β-strands and two α-helices sequentially appearing in the order β1, α1, α2, β2, β3, β4, and β5, the β-strands being arranged in two anti-parallel β-sheets, one β-sheet composed of β1 and β5, the other β-sheet composed of β2, β3, and β4, and   at least two disulfide bridges, one disulfide bridge connecting α1 and β5 and one disulfide bridge connecting β3 and a polypeptide segment connecting β4 and β5; and   the randomized CTLD loop region consisting of two loop polypeptide segments, loop segment A (LSA) connecting β2 and β3, and loop segment B (LSB) connecting β3 and β4, wherein the amino acid sequence of LSA and/or LSB is randomized from the amino acid sequence of a wildtype CTLD loop region by random amino acid substitution, deletion, insertion, or any combination thereof of the wildtype CTLD loop sequence.   
     
     
         78 . The combinatorial library of  claim 77 , wherein the CTLD is selected from a human tetranectin (hTN), mannose binding protein (MBP), surfactant protein D (SP-D), LY49A NK receptor domain (LY49A), asidoglycoprotein receptor (Hl-ASR), mouse macrophage receptor (MMR-4), Factor 1X/X binding protein A (IX-A), Factor 1X/X binding protein B (IX-B), lithostatin (Lit), tunicate C-type lectin (TU14) CTLD, and mouse tetranectin (mTN) CTLD. 
     
     
         79 . The combinatorial library of  claim 77 , wherein the polypeptides further comprise N-terminal and/or C-terminal extensions of the CTLD. 
     
     
         80 . The combinatorial library of  claim 79 , wherein the N-terminal and/or C-terminal extensions contain effector, enzyme, further binding and/or multimerizing functions. 
     
     
         81 . The combinatorial library of  claim 79 , wherein the N-terminal and/or C-terminal extensions are the non-CTLD-portions of a native C-type lectin-like protein or a C-type lectin or a C-type lectin lacking a functional transmembrane domain. 
     
     
         82 . The combinatorial library of  claim 77 , wherein the amino acid residues differ between different members of the ensemble of polypeptides in at least at two amino acid sequence positions in the randomized loop region. 
     
     
         83 . The combinatorial library of  claim 82 , wherein at least three of the amino acid sequence positions of Loop Segment A are randomized. 
     
     
         84 . The combinatorial library of  claim 82 , wherein at least two of the amino acid sequence positions of Loop Segment B are randomized. 
     
     
         85 . The combinatorial library of  claim 77 , wherein the amino acid residues differ between different members of the ensemble of polypeptides at least at one amino acid sequence position in the Loop Segment A and at least at one amino acid sequence position in Loop Segment B. 
     
     
         86 . The combinatorial library of  claim 77 , wherein the amino acid residues differ between different members of the ensemble of polypeptides at any one or more sequence positions in the loop region corresponding to
 amino acid residues 72-107 and 114-117 of SEQ ID NO: 276;   amino acid residues 66-99 and 105-107 of SEQ ID NO: 277;   amino acid residues 69-102 and 108-110 of SEQ ID NO: 278;   amino acid residues 72-93 and 99-100 of SEQ ID NO: 279;   amino acid residues 62-101 and 107-109 of SEQ ID NO: 280;   amino acid residues 77-111 and 117-121 of SEQ ID NO: 281;   amino acid residues 71-100 and 105-112of SEQ ID NO: 282;   amino acid residues 68-94 and 99-104 of SEQ ID NO: 283;   amino acid residues 68-103 and 111-117 of SEQ ID NO: 284;   amino acid residues 54-94 and 100-103 of SEQ ID NO: 285; and   amino acid residues 115-151 and 158-161 of SEQ ID NO: 289.   
     
     
         87 . The combinatorial library of  claim 86 , wherein the amino acid residues differ between different members of the ensemble of polypeptides at any one or more sequence positions in the loop region corresponding to amino acid residues 72-79, 81-85, 91-99, 101-107, and 114-117 of SEQ ID NO: 276. 
     
     
         88 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any of the sequence positions corresponding to 73-78, 93-98, 102-105, and 114-117 of SEQ ID NO: 276. 
     
     
         89 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any of the sequence positions corresponding to 73-75, 77-78, and 102-105 of SEQ ID NO: 276. 
     
     
         90 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any of the sequence positions corresponding to 93-98 and 102-105 of SEQ ID NO: 276. 
     
     
         91 . The combinatorial library of  claim 86 , wherein the amino acid residues further differ at the sequence position corresponding to120 of SEQ ID NO: 276. 
     
     
         92 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any of the sequence positions corresponding to 93-98 and 114-117 of SEQ ID NO: 276. 
     
     
         93 . The combinatorial library of  claim 88 , wherein the amino acid residues further differ at any of the sequence positions corresponding to 112, 113, and 118 of SEQ ID NO: 276. 
     
     
         94 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any of the sequence positions corresponding to 94-97 and 114-116 of SEQ ID NO: 276. 
     
     
         95 . The combinatorial library of  claim 94 , wherein the amino acid residues differ at any of the sequence positions corresponding to 73-75, 77-78, and 104-105 of SEQ ID NO: 276. 
     
     
         96 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any one or more sequence positions in the loop region corresponding to amino acid residues 66-73, 75-79, 85-90, 92-99, and 105-107 of SEQ ID NO: 277. 
     
     
         97 . The combinatorial library of  claim 86 , wherein the amino acid residues differ at any one or more sequence positions in the loop region corresponding to amino acid residues 69-76, 78-82, 88-93, 95-102, and 108-110 of SEQ ID NO: 278. 
     
     
         98 . The combinatorial library of  claim 77 , wherein 1-10 amino acid residues are substituted, deleted, or inserted in any one or more of the α-helices, β-strands, and connecting segments. 
     
     
         99 . The combinatorial library of  claim 77 , wherein the 1-10 amino acid residues are substituted, deleted, or inserted in any one or more of the β2, β3, and β4-strands. 
     
     
         100 . The combinatorial library of  claim 77 , wherein the polypeptide sequence outside of the loop region is at least 95% identical to the amino acid sequence outside the loop region of one of SEQ ID NO:276, 277 and 278. 
     
     
         101 . A combinatorial library comprising an ensemble of polypeptides comprising an amino acid sequence at least 95% identical to amino acids 1-71, and 114-117 of SEQ ID NO:276 wherein one or more of amino acids 72-79, 81-85, 91-99, 101-107, and 114-117 are randomized. 
     
     
         102 . A nucleic acid library comprising a multitude of nucleic acids encoding variant C-type lectin-like domain (CTLD) polypeptides having the scaffold structure of a CTLD polypeptide and a randomized CTLD loop region, the CTLD scaffold comprising the following structural elements:
 five β-strands and two α-helices sequentially appearing in the order β1, α1, α2, β2, β3, β4, and β5, the (β-strands being arranged in two anti-parallel (β-sheets, one (3-sheet composed of β1 and β5, the other (3-sheet composed of β2, β3, and β4, and   at least two disulfide bridges, one disulfide bridge connecting α1 and β5 and one disulfide bridge connecting β3 and a polypeptide segment connecting β4 and β5; and   the randomized CTLD loop region consisting of two loop polypeptide segments, loop segment A (LSA) connecting β2 and β3, and loop segment B (LSB) connecting β3 and β4, wherein the amino acid sequence of LSA and/or LSB is randomized from the amino acid sequence of a wildtype CTLD loop region by random amino acid substitution, deletion, insertion, or any combination thereof of the wildtype CTLD loop sequence.   
     
     
         103 . The library of  claim 102 , wherein the CTLD loop region is randomized by substituting the portion of the nucleic acid molecules encoding some or all of the loop regions with a nucleic acid fragment randomly selected from a multitude of nucleic acid fragments. 
     
     
         104 . The combinatorial library of  claim 103 , wherein the amino acid residues differ between different members of the ensemble of polypeptides in at least at two amino acid sequence positions in the randomized loop region. 
     
     
         105 . The combinatorial library of  claim 103 , wherein at least three of the amino acid sequence positions of Loop Segment A are randomized. 
     
     
         106 . The combinatorial library of  claim 103 , wherein at least two of the amino acid sequence positions of Loop Segment B are randomized. 
     
     
         107 . The library of  claim 102 , wherein the nucleotide sequence encoding the polypeptide sequence outside of the loop region is altered to facilitate the excision of part or all of the loop region and the insertion of an altered loop polypeptide sequence while the scaffold structureof the CTLD is substantially maintained. 
     
     
         108 . A method of preparing the combinatorial library based upon a CTLD scaffold structure, the method comprising:
 (a) inserting in a suitable vector a nucleic acid encoding variant C-type lectin-like domain (CTLD) polypeptide having the scaffold structure of a CTLD polypeptide and a randomized CTLD loop region, the CTLD scaffold comprising the following structural elements:   five β-strands and two α-helices sequentially appearing in the order β1, α1, α2, β2, β3, β4, and β5, the (β-strands being arranged in two anti-parallel (β-sheets, one (β-sheet composed of β1 and β5, the other (β-sheet composed of β2, β3, and β4, and   at least two disulfide bridges, one disulfide bridge connecting α1 and β5 and one disulfide bridge connecting β3 and a polypeptide segment connecting β4 and β5; and   the randomized CTLD loop region consisting of two loop polypeptide segments, loop segment A (LSA) connecting β2 and β3, and loop segment B (LSB) connecting β3 and β4, wherein the amino acid sequence of LSA and/or LSB is randomized from the amino acid sequence of a wildtype CTLD loop region by random amino acid substitution, deletion, insertion, or any combination thereof of the wildtype CTLD loop sequence,   (b) optionally introducing restriction endonuclease recognition sites, the recognition sites being properly located in the sequence at or close to the ends of the sequence encoding the loop region of the CTLD or part thereof,   (c) excising the DNA fragment encoding the loop region or part thereof using restriction endonucleases;   (d) ligating randomized mixtures of DNA fragments into the loop region of the restricted vector, and   
       inducing the vector to express randomized polypeptides having the scaffold structure of the CTLD and a randomized loop region in a suitable medium.