Use of lambda-gam protein in ribosomal display technology

Abstract

Methods and systems for increasing the stability of a nucleic acid template that encodes a protein of interest in a cell free translation system or a ribosomal display reaction system are described. In some embodiments, the nucleic acid template is an RNA or mRNA. The stability of the RNA template is increased by adding the bacteriophage lambda protein Gam to the cell free extract used in the translation system. The addition of Gam protein increases the longevity of the reaction system, thereby increasing the efficiency of the ribosomal display reaction system.

1 .- 15 . canceled 
     
     
         16 . A method of selecting for a protein of interest using a ribosomal display reaction system, the method comprising:
 i) combining a library of nucleic acid members, each member encoding a prospective protein of interest having a primary amino acid different from the other proteins encoded by the other nucleic acid members, and where the members lack an operable stop codon, with a cell free translation system to yield a ribosomal display reaction system containing a mixture of different complexes, each complex comprising a different mRNA encoding the prospective protein of interest, a ribosome, and a prospective protein of interest, wherein the system has a concentration of λGam protein sufficient to stabilize the mRNA encoding the protein of interest; and,   ii) selecting proteins of interest from the ribosomal display reaction system by binding the complexes to an immobilized binding pair ligand that binds to proteins of interest.   
     
     
         17 . The method of  claim 16 , wherein the λGam protein is the S form. 
     
     
         18 . The method of  claim 16 , where the λGam protein is present at a concentration of 25-60 μg/ml of extract. 
     
     
         19 . The method of  claim 16 , where the protein of interest is an antibody. 
     
     
         20 . The method of  claim 16 , where the protein of interest is a single chain antibody. 
     
     
         21 . The method of  claim 16 , where the ribosomal display reaction system comprises two or more nucleic acid templates, wherein each template encodes a protein of interest having a different primary amino acid sequence from the other templates. 
     
     
         22 . The method of  claim 16 , where the method further comprises the step of capturing the protein of interest to a binding pair member that is immobilized to a support substrate. 
     
     
         23 . The method of  claim 22 , where the capturing step is repeated to enrich for optimal binding. 
     
     
         24 . The method of  claim 16 , where the nucleic acid member is RNA and it is transcribed from DNA. 
     
     
         25 . The method of  claim 16 , where the mRNA of the complexes of step (ii) are amplified and reintroduced into a second cell free translation system to yield a second RDRS enriched for proteins of interest able to bind to the immobilized binding pair ligand. 
     
     
         26 . The method of  claim 16 , wherein the λGam protein comprises one or more amino acid substitutions that increase or further increase the stability of the mRNA encoding the protein of interest, or comprises amino acid substitutions that do not decrease the stability of the mRNA encoding the protein of interest, as compared to a control, unmodified or prototype Gam protein. 
     
     
         27 . The method of  claim 26 , wherein the λGam protein comprises amino acid substitutions N2>S, A3>T, Y4>W or C, Y5>L or P, Q7>P, R9>C, E11>K, Al2>E, 14S>A, 16A>T or S, 20Q>R, 21Q>K, 22L>I, A23>V or T, 27K>T or N, 29A>T, 33D>E, 34D>N, 35M>L, I39>L, 51H>Q, N or S, 55H>C, 60K>Q, 61S>A or T, 63T>I, S, or A, 84V>A, 92V>A or I, and 93D>N or Y, of SEQ ID NO:1. 
     
     
         28 . The method of  claim 16 , wherein the λGam protein binds polyclonal antibodies that bind the prototype protein of SEQ ID NO:1. 
     
     
         29 . The method of  claim 16 , wherein the λGam protein is encoded by the lambda gam (gamma) gene (Gene ID: 2703509). 
     
     
         30 . The method of  claim 17 , wherein the λGam protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1.