Structure-activity relationships

Abstract

The present disclosure relates to compositions and methods for screening a plurality of polypeptide variants.

1 . A plurality of addressable polypeptides, a portion of each polypeptide having known amino acid sequence, wherein each polypeptide reacts with a substrate to produce a product, and wherein at least two members of the plurality are related to a parent polypeptide and react with different substrates. 
   
   
       2 . A plurality of addressable polypeptides, a portion of each polypeptide having known amino acid sequence, wherein each polypeptide reacts with a substrate to produce a product, and wherein at least two members of the plurality are related to a parent polypeptide and react with differing levels of activity upon a given substrate. 
   
   
       3 . The plurality of  claim 1  in which each member acts on a different substrate. 
   
   
       4 . The plurality of  claim 1  or  claim 2  in which some members act on one substrate and other members act on at least one other substrate. 
   
   
       5 . The plurality of  claim 1  or  claim 2  in which at least two members of the plurality differ with respect to one or more of the following properties: substrate specificity, chemoselectivity, regioselectivity, stereoselectivity, stereospecificity, ligand specificity, receptor agonism, receptor antagonism, conversion of a cofactor. 
   
   
       6 . The plurality of  claim 4  in which the members of the plurality that act on the same substrate differ with respect to one or more of the following properties: rate of product formation, percent conversion of a substrate to a product, or percent conversion of a cofactor. 
   
   
       7 . The plurality of  claim 1  or  claim 2  in which at least two members are operable over a pH range from about pH 5 to about pH 10. 
   
   
       8 . The plurality of  claim 1  or  claim 2  in which at least two members are operable over a range of temperatures ranging from about 4° C. to about 50° C. 
   
   
       9 . The plurality of  claim 1  or  claim 2  in which at least two members are operable in a solution containing from about 10 to about 50% organic solvent. 
   
   
       10 . The plurality of  claim 1  or  claim 2  in which each member of the plurality is an evolvant of the same parent enzyme and contains at least one mutation as compared to the parent enzyme. 
   
   
       11 . The plurality of  claim 1  or  claim 2  in which the plurality comprises subsets of members and each subset comprises evolvants from a separate parent enzyme. 
   
   
       12 . A plurality of pluralities in which each plurality acts on a different class of substrate. 
   
   
       13 . A plurality of enzymes made by a process of:
 (i) generating variants of at least one parent enzyme, wherein at least a portion of the amino acid sequence of each variant is known, and wherein each variant comprises at least one mutation known to affect reactivity with a substrate to produce a product; and   (ii) selecting two or more variants wherein at least two of the selected variants react with different substrates.   
   
   
       14 . A plurality of enzymes made by a process of:
 (i) generating variants of at least one parent enzyme, wherein at least a portion of the amino acid sequence of each variant is known, and wherein each variant comprises at least one mutation known to affect reactivity with a substrate to produce a product; and   (ii) selecting two or more variants wherein at least two of the selected variants react with differing levels of activity upon a given substrate.   
   
   
       15 . The plurality of  claim 13  in which each of the selected variants acts on a different substrate. 
   
   
       16 . The plurality of  claim 13  or  14  in which some members of the plurality act on one substrate and other members of the plurality act on at least one other substrate. 
   
   
       17 . The plurality of  claim 13  or  14  in which at least two members of the plurality differ with respect to one or more of the following properties: substrate specificity, chemoselectivity, regioselectivity, stereoselectivity, stereospecificity, ligand specificity, receptor agonism, receptor antagonism, conversion of a cofactor. 
   
   
       18 . The plurality of  claim 16  in which the members of the plurality that act on the same substrate differ with respect to one or more of the following properties: rate of product formation, percent conversion of a substrate to a product, or percent conversion of a cofactor. 
   
   
       19 . The plurality of  claim 13  or  14  in which at least two members of the plurality are operable over a pH range from about pH 5 to about pH 10. 
   
   
       20 . The plurality of  claim 13  or  14  in which at least two members of the plurality are operable over a range of temperatures ranging from about 4° C. to about 50° C. 
   
   
       21 . The plurality of  claim 13  or  14  in which at least two members of the plurality are operable in a solution containing from about 10 to about 50% organic solvent. 
   
   
       22 . The plurality of  claim 13  or  14  in which each member of the plurality is an evolvant of the same ancestor enzyme and contains at least one mutation as compared to the ancestor enzyme. 
   
   
       23 . The plurality of  claim 13  or  14  in which the plurality comprises subsets of members and each subset comprises evolvants from a separate ancestor enzyme. 
   
   
       24 . The plurality of  claim 13  or  14  in which the selected variants are placed onto a surface. 
   
   
       25 . The plurality of  claim 13  or  14  in which the surface comprises a microtiter plate. 
   
   
       26 . The plurality of  claim 13  or  14  in which the surface comprises a set of 2 to 4 microtiter plates. 
   
   
       27 . The plurality of  claim 13  or  14  in which the surface comprises a microchip. 
   
   
       28 . The plurality of  claim 13  or  14  in which the surface comprises glass. 
   
   
       29 . The plurality of  claim 13  or  14  in which the surface comprises plastic. 
   
   
       30 . The plurality of  claim 13  or  14  in which the variants are expressed from polynucleotides. 
   
   
       31 . The plurality of  claim 13  or  14  in which the variants are chemically synthesized. 
   
   
       32 . A plurality of addressable polypeptide variants made by a process of:
 (i) generating a plurality of polypeptide variants related to a parent polypeptide, each variant having a different amino acid sequence, wherein each variant comprises at least one mutation that enhances reactivity with a ligand as compared to the reactivity of the parent polypeptide with said ligand; and wherein at least two variants of the plurality react with different ligands; and   (ii) assigning an address to each of the variants.   
   
   
       33 . A plurality of addressable polypeptide variants made by a process of:
 (i) generating a plurality of polypeptide variants related to a parent polypeptide, each variant having a different amino acid sequence, wherein each variant comprises at least one mutation that enhances reactivity with a ligand as compared to the reactivity of the parent polypeptide with said ligand, and wherein at least two variants of the plurality react with differing levels of activity upon a given ligand; and   (ii) assigning an address to each of the variants.   
   
   
       34 . A method useful for identifying at least one polypeptide having a desired activity, comprising:
 (i) screening a plurality of polypeptide variants against a ligand of interest, wherein each variant reacts with at least one ligand to produce a detectable signal, and wherein at least two members of the plurality are related to a parent polypeptide and react with different ligands; and   (ii) identifying at least one variant that produces a signal upon reaction with the ligand of interest.   
   
   
       35 . A method useful for identifying at least one polypeptide having a desired activity, comprising:
 (i) screening a plurality of polypeptide variants against a ligand of interest, wherein each variant reacts with at least one ligand to produce a detectable signal, and wherein at least two members of the plurality are related to a parent polypeptide and react with differing levels of activity upon a given ligand; and   (ii) identifying at least one variant that produces a signal upon reaction with the ligand of interest.   
   
   
       36 . The method of  claim 34  or  35 , further comprising:
 (iii) identifying at least a second variant that produces a signal upon reaction with the ligand of interest;   (iv) postulating a sequence-activity relationship between the amino acid residues within the identified variants of  claim 34  or  35  and the ability of each variant to produce a detectable signal;   (v) generating a second plurality of polypeptide variants based on the sequence-activity relationship;   (vi) screening the second plurality against the ligand of interest; and   (vii) identifying at least one variant from the second plurality that produces an enhanced signal of interest as compared to the identified variants of  claim 34  or  35 .   
   
   
       37 . The method of  claim 34  or  35 , in which the identity of the ligand of interest is unknown. 
   
   
       38 . The method of  claim 36 , in which the identity of the ligand of interest is unknown. 
   
   
       39 . The method of  claim 34  or  35 , in which each variant is an enzyme and the ligand of interest is a candidate substrate for the enzyme. 
   
   
       40 . The method of  claim 34  or  35 , in which each variant is a receptor and the ligand of interest is a candidate ligand for the receptor. 
   
   
       41 . The method of  claim 34  or  35 , in which two or more members of the plurality differ in the kind of signal produced. 
   
   
       42 . The method of  claim 34  or  35 , in which the kind of signal produced is selected from a signal indicating substrate specificity, chemoselectivity, regioselectivity, stereoselectivity, stereospecificity, ligand specificity, receptor agonism, receptor antagonism, conversion of a cofactor, or any combination thereof. 
   
   
       43 . The method of  claim 34  or  35 , in which two or more members of the plurality differ in the level of signal produced. 
   
   
       44 . The method of  claim 43  in which the difference in level is a difference in rate of product formation, percent conversion of a substrate to a product, or percent conversion of a cofactor. 
   
   
       45 . A host cell which produces the at least one variant identified in the method of  claim 34  or  claim 35 . 
   
   
       46 . A plurality of host cell colonies, wherein each colony expresses a member of the plurality of  claim 34  or  35 . 
   
   
       47 . A method of making a plurality of addressable polypeptide variants, comprising:
 (i) generating a plurality of polypeptide variants related to a parent polypeptide, a portion of each variant having known amino acid sequence, wherein each variant comprises at least one mutation that enhances reactivity with a substrate as compared to the reactivity of the parent polypeptide with said substrate, and wherein at least two members of the plurality react with different substrates; and   (ii) assigning an address to each of the variants.   
   
   
       48 . A method of making a plurality of addressable polypeptide variants, comprising:
 (i) generating a plurality of polypeptide variants related to a parent polypeptide, a portion of each variant having known amino acid sequence, wherein each variant comprises at least one mutation that enhances reactivity with a substrate as compared to the reactivity of the parent polypeptide with said substrate, and wherein at least two members of the plurality react with differing levels of activity upon a given substrate; and   (ii) assigning an address to each of the variants.   
   
   
       49 . The method of  claim 47  in which each member of the plurality acts on a different substrate. 
   
   
       50 . The method of  claim 47  or  claim 48  in which some members act on one substrate and other members act on at least one other substrate. 
   
   
       51 . The method of  claim 47  or  claim 48  in which at least two members of the plurality differ with respect to one or more of the following properties: substrate specificity, chemoselectivity, regioselectivity, stereoselectivity, stereospecificity, ligand specificity, receptor agonism, receptor antagonism, conversion of a cofactor. 
   
   
       52 . The method of  claim 50  in which the members of the plurality that act on the same substrate differ with respect to one or more of the following properties: rate of product formation, percent conversion of a substrate to a product, or percent conversion of a cofactor. 
   
   
       53 . The method of  claim 47  or  claim 48  in which at least two members of the plurality are operable over a pH range from about pH 5 to about pH 10. 
   
   
       54 . The method of  claim 47  or  claim 48  in which at least two members of the plurality are operable over a range of temperatures ranging from about 4° C. to about 50° C. 
   
   
       55 . The method of  claim 47  or  claim 48  in which at least two members of the plurality are operable in a solution containing from about 10 to about 50% organic solvent. 
   
   
       56 . The method of  claim 47  or  claim 48  in which each member of the plurality is an evolvant of the same parent enzyme and contains at least one mutation as compared to the parent enzyme. 
   
   
       57 . The method of  claim 47  or  claim 48  in which the plurality comprises subsets of members and each subset comprises evolvants from a separate parent enzyme. 
   
   
       58 . The method of  claim 47  or  claim 48  in which each member of each plurality is an enzyme and the at least one ligand is a substrate for the enzyme. 
   
   
       59 . The method of  claim 47  or  claim 48  in which each member of each plurality is a receptor and the at least one ligand is a ligand for the receptor. 
   
   
       60 . A method of making a plurality of addressable pluralities, comprising:
 (i) generating a first and a second addressable plurality of polypeptide variants, wherein the members of the first plurality are related to one parent polypeptide and the members of the second plurality are related to a second parent polypeptide, wherein at least a portion of the amino acid sequence of each variant is known, wherein each member of each plurality reacts with a substrate to produce a product, and wherein at least one member of the first plurality and at least one member of the second plurality react with different substrates.   
   
   
       61 . A kit for identifying at least one polypeptide having a desired activity, comprising:
 (i) at least one plurality of addressable polypeptides, wherein each member of each plurality reacts with at least one ligand to produce a detectable signal, and wherein at least two members of each plurality are related to a parent polypeptide and react with different ligands; and   (ii) instructions for use.   
   
   
       62 . A kit for identifying at least one polypeptide having a desired activity, comprising:
 (i) at least one plurality of addressable polypeptides, wherein each member of each plurality reacts with at least one ligand to produce a detectable signal, and wherein at least two members of each plurality are related to a parent polypeptide and react with differing levels of activity upon a given ligands; and   (ii) instructions for use.