Method for Producing and Identifying Soluble Protein Domains

Abstract

Methods for producing and identifying fragments of proteins, and more particularly to methods for generating and identifying soluble protein domains are disclosed based on a method for generating a library of nucleic acid fragments from nucleic acid encoding a desired polypeptide, and more especially a library of essentially, randomly sampled fragments of coding DNA sequence predominantly of defined size range and a method for selecting cloned gene fragments from the library that encode soluble protein domains.

1 - 40 . (canceled) 
     
     
         41 . A method of producing a library of nucleic acid fragments, the nucleic acid fragments encoding one or more portions of a polypeptide, and identifying fragments in the library encoding soluble protein domains, the method comprising:
 amplifying a nucleic acid sequence encoding the polypeptide in the presence of a non-native nucleotide so that the non-native nucleotide is incorporated into the amplified product nucleic acid sequence at a frequency related to the relative amounts of the non-native nucleotide and its corresponding native nucleotide, if present;   contacting the product nucleic acid sequence with one or more reagents capable of recognising the presence of the non-native nucleotide and cleaving the product nucleic acid sequence or excising the non-native nucleotide, thereby producing nucleic acid sequences encoding fragments of the polypeptide;   expressing a library of the nucleic acid fragments to produce the protein domains encoded by the fragments, wherein the protein domains are expressed as fusions with an affinity tag; and   separating soluble proteins using the affinity tag.   
     
     
         42 . A method for producing a library of nucleic acid fragments, the nucleic acid fragments encoding one or more portions of a polypeptide, the method comprising: amplifying a nucleic acid sequence encoding the polypeptide in the presence of a non-native nucleotide so that the non-native nucleotide is incorporated into an amplified product nucleic acid sequence at a frequency related to the relative amounts of the non-native nucleotide and its corresponding native nucleotide, if present;
 contacting the product nucleic acid sequence with one or more reagents capable of recognising the presence of the non-native nucleotide and cleaving the product nucleic acid sequence or excising the non-native nucleotide, thereby producing nucleic acid sequences encoding fragments of the polypeptide.   
     
     
         43 . The method of  claim 41  or  claim 42 , wherein the step of amplifying the nucleic acid sequence is carried out using PCR using a non-native deoxynucleotide, either alone or in a mixture of the non-native and native nucleotide. 
     
     
         44 . The method of  claim 43 , wherein the non-native nucleotide is uracil or 3-methyl adenine. 
     
     
         45 . The method of any one of the preceding claims, wherein the nucleic acid sequence is present in a cDNA library, a RNA library or a sample of genomic DNA. 
     
     
         46 . The method of any one of the preceding claims, wherein the nucleic acid fragments of the polypeptide are between 200 and 1200 nucleotides in length. 
     
     
         47 . The method of any one of the preceding claims, wherein the nucleic acid sequence is sampled on average about every second nucleotide to produce the nucleic acid fragments. 
     
     
         48 . The method of any one of the preceding claims, wherein the reagent capable of recognising and cleaving the product nucleic acid sequence at the non-native nucleotide is an enzyme which can recognise the presence of the non native nucleotide and cleave the nucleic acid sequence at or around the modified nucleic acid sequence. 
     
     
         49 . The method of  claim 48 , wherein the enzyme is a DNA glycosylase or an endonuclease. 
     
     
         50 . The method of any one of the preceding claims, wherein the non-native nucleotide is deoxyuracil and the enzyme is apurinic/apyrimidinic endonuclease (APE), catalysed by uracil-DNA glycosylase (UDG). 
     
     
         51 . The method of any one of the preceding claims, further comprising amplifying the nucleic acid fragments. 
     
     
         52 . The method of any one of the preceding claims, wherein in the library of protein domains, the protein domains comprise a constant portion and a portion sampled by the amplifying and contacting steps. 
     
     
         53 . The method of any one of the preceding claims, further comprising ligating the nucleic acid fragments into expression vector(s). 
     
     
         54 . The method of  claim 53 , further comprising transforming host cells with the expression vectors to produce a library of host cells capable of expressing fragments of the polypeptide. 
     
     
         55 . The method of  claim 54 , further comprising expressing the nucleic acid sequences encoding fragments of the polypeptide and optionally isolating the polypeptide fragments thus produced. 
     
     
         56 . A method of identifying soluble protein domains, the method comprising:
 expressing a library of nucleic acid fragments to produce the protein domains encoded by the fragments, wherein the protein domains are expressed as fusions with an affinity tag; and   separating soluble proteins using the affinity tag.   
     
     
         57 . The method of any one of the preceding claims, wherein the polypeptide fragments are expressed to include a protease cleavage site. 
     
     
         58 . The method of any one of the preceding claims, wherein the polypeptide fragments are expressed to include an affinity tag. 
     
     
         59 . The method of  claim 58 , wherein the affinity tag is a peptide which is less than 15 amino acids in length. 
     
     
         60 . The method of  claim 58  or  claim 59 , wherein the affinity tag is fused to the C-terminus of the protein fragments. 
     
     
         61 . The method of any one of  claims 58  to  60 , wherein the affinity tag is polyhistidine, a Flag or Glu epitope, a S-tag, calmodulin binding peptide or ribonuclease S. 
     
     
         62 . The method of  claim 61 , wherein the affinity tag is a His 6 -tag. 
     
     
         63 . The method of any one of  claims 54  to  62 , further comprising releasing the soluble protein domains from the cells. 
     
     
         64 . The method of  claim 63 , wherein the step of releasing the protein is carried out under non-denaturing conditions. 
     
     
         65 . The method of  claim 64 , wherein the non-denaturing condition comprises the use of enzymes or non-denaturing detergents. 
     
     
         66 . The method of any one of  claims 54  to  65 , further comprising filtering out unbroken cells, cell debris and insoluble material. 
     
     
         67 . The method of any one of  claims 54  to  66 , further comprising clarifying the lysates by centrifugation. 
     
     
         68 . The method of any one of  claims 54  to  67 , further comprising purifying cells transformed with different proteins in parallel by affinity chromatography. 
     
     
         69 . The method of any one of the preceding claims, wherein the step of separating the soluble protein domains is carried out by contacting the library of protein domains with a solid phase having a binding partner of the affinity tag immobilised thereon. 
     
     
         70 . The method of  claim 69 , wherein the binding partner is:
 (a) metal ions such as Ni 2+  or Co 2+  for binding a polyhistidine affinity tag; or   (b) anti-Flag antibodies for binding a Flag or Glu epitope affinity tag; or   (c) streptavidin for binding a S-tag affinity tag; or   (d) calmodulin in the presence of Ca 2+  for binding a calmodulin binding peptide affinity tag; or   (e) aporibonuclease S for binding a ribonuclease S affinity tag.   
     
     
         71 . The method of any one of the preceding claims, further comprising assaying for the presence of soluble protein fragments. 
     
     
         72 . The method of  claim 71 , wherein the step of assaying is carried out using anti-tag ELISA, SDS-PAGE or LC-ESI-MS. 
     
     
         73 . The method of  claim 71  or  claim 72 , wherein the step of assaying for the soluble protein domains comprises quantifying the protein expression level of one or more or the protein domains. 
     
     
         74 . The method of any one of the preceding claims, further comprising identifying or sequencing the soluble proteins. 
     
     
         75 . The method of any one of the preceding claims, further comprising contacting the library of fragments or domains with: 
       (a) one or more candidate compounds to determine whether the candidate compound binds to and/or modulates an activity of a protein fragment or domain present in the library; and/or 
       (b) one or more test proteins to determine whether a protein fragment or domain present in the library binds to and/or modulates an activity of the test protein. 
     
     
         76 . The method of any one of the preceding claims, further comprising contacting two or more libraries of soluble protein fragments or domains to determine whether binding or modulation of activity occurs between the protein fragments or domains present in the libraries. 
     
     
         77 . The method of  claim 76 , wherein the method is employed to determine which portions of the proteins used to construct the libraries are involved in binding and biological activity. 
     
     
         78 . The method of any one of  claims 75  to  77 , wherein the method is used to determine binding between a ligand and a receptor, an enzyme and a substrate, an antibody and an antigen, or a small molecule and a protein. 
     
     
         79 . A library produced by the method of any one of the preceding claims. 
     
     
         80 . The library of  claim 79 , which is: 
       (a) a library of nucleic acid fragments of a parent nucleic acid sequence, wherein the nucleic acid fragments have a size range between 200 and 1200 nucleotides in length and are preferably sampled from the parent nucleic acid sequence on average about every second nucleotide; or 
       (b) a library of expression vectors which comprise a plurality of the nucleic acid fragments as set out in (a), wherein each fragment is ligated to a nucleic acid sequence encoding an affinity tag and optionally one or more further sequences to direct the expression of the nucleic acid fragment and the affinity tag; or,
 (c) a library of host cells transformed with the expression vectors as defined in (b); or 
 (d) a library of polypeptide fragments produced by expressing the nucleic acid sequences set out in step (a), wherein each polypeptide is coupled to an affinity tag.