Ribosome display

Abstract

The use in a ribosome display system for selection of a specific binding pair member (e.g. antibody molecule) able to bind a complementary specific binding pair member (e.g. antigen) of encapsidating specific binding member/ribosome complexes in a viral coat, optionally in combination with incorporation of an Midvariant RNA template and optionally one or more other improvements selected from: a glycine-serine tether, protein disulphide isomerase, protein disulphide isomerase in combination with oxidised and reduced glutathione at a ratio of between 1:1 and 10:1, addition of oxidised and reduced glutathione at a ratio of between 1:1 and 10:1 after 30 minutes of in vitro translation; blocking with heparin during selection.

1 . A method of obtaining a specific binding pair (sbp) member that binds a complementary sbp member of interest, the method comprising: 
 (a) providing mRNA molecules, each mRNA molecule comprising a nucleotide sequence encoding a specific binding pair member and lacking an in-frame stop codon;    (b) incubating the mRNA molecules under conditions for ribosome translation of the mRNA molecules to produce encoded specific binding pair member, whereby complexes each comprising ribosome, mRNA and encoded specific binding pair member displayed on the ribosome are formed;    (c) bringing the complexes into contact with the complementary sbp member of interest, and selecting one or more complexes displaying specific binding pair member able to bind the complementary sbp member of interest under the conditions of the selection;    wherein the mRNA molecules are incubated with prokaryotic ribosomes in a prokaryotic ribosome display system or are incubated with eukaryotic ribosomes in a eukaryotic ribosome display system;    and wherein    mRNA molecules for incubation in the translation system are provided by means of RT-PCR reactions in which at least one RT-PCR primer is a mutagenic primer encoding a diversity of different sequences for inclusion in a defined region of the nucleotide sequence encoding a specific binding pair member.    
     
     
         2 . The method according to  claim 1  wherein the mRNA molecules incorporate a Midvariant (MDV) RNA template enabling replication by Qβ replicase.  
     
     
         3 . The method according to  claim 1  wherein a gly-ser tether is fused C-terminally to specific binding pair member.  
     
     
         4 . The method according to  claim 3  wherein the gly-ser tether comprises 24 glycine-serine units.  
     
     
         5 . The method according to  claim 1  wherein oxidised and reduced glutathione is added at a ratio of between 1:1 and 10:1 after 30 minutes of ribosome translation.  
     
     
         6 . The method according to  claim 1  wherein protein disulphide isomerase (PDI) is employed in the incubation conditions, along with oxidised and reduced glutathione at a ratio of 1:1 and 10:1.  
     
     
         7 . The method according to  claim 1  wherein the translation system is eukaryotic and protein disulphide isomerase (PDI) is employed in the incubation conditions.  
     
     
         8 . The method according to  claim 1  comprising selecting for complexes comprising a specific binding member able to bind complementary specific binding member of interest, while blocking unspecific selection using heparin.  
     
     
         9 . The method according to  claim 1  further comprising retrieving mRNA from a complex selected in step (c).  
     
     
         10 . The method according to  claim 9  comprising employing a mutagenic primer in RT-PCR generation of a DNA copy of the ribosome display library prior to a further round of selection.  
     
     
         11 . The method according to  claim 9  wherein mRNA retrieved from a selected complex displaying a specific binding pair member (a “selected specific binding pair member”) is amplified and copied into DNA encoding the selected specific binding pair member.  
     
     
         12 . The method according to  claim 11  wherein the DNA is provided in an expression system for production of a product, which product is the selected specific binding pair member or a polypeptide chain of the selected specific binding pair member.  
     
     
         13 . The method according to  claim 12  further comprising isolating or purifying the product.  
     
     
         14 . The method according to  claim 13  further comprising formulating the product into a composition comprising at least one additional component.  
     
     
         15 . The method according to  claim 14  wherein DNA encoding the selected specific binding pair member or a polypeptide chain of the selected specific binding pair member is provided within a nucleotide sequence to provide a nucleotide sequence encoding a fusion protein comprising the selected specific binding pair member, or a polypeptide chain of the selected specific binding pair member, fused to additional amino acids.  
     
     
         16 . The method according to  claim 15  wherein the selected specific binding pair member comprises an antibody VH and/or antibody VL domain and the additional amino acids comprise an antibody constant domain.  
     
     
         17 . The method according to  claim 15  wherein DNA comprising said nucleotide sequence encoding said fusion protein is provided in an expression system for production of a product, which product is the fusion protein.  
     
     
         18 . The method according to  claim 17  further comprising isolating or purifying the product.  
     
     
         19 . The method according to  claim 18  further comprising formulating the product into a composition comprising at least one additional component.  
     
     
         20 . The method according to  claim 11  wherein DNA encoding the selected specific binding pair member or a polypeptide chain of the selected specific binding pair member is mutated to encode a polypeptide that comprises an amino acid sequence that differs from the selected specific binding pair member or polypeptide chain of the selected specific binding pair member.  
     
     
         21 . The method according to  claim 20  wherein mutated DNA encoding said polypeptide is provided in an expression system for production of a product, which product is said polypeptide.  
     
     
         22 . The method according to  claim 21  further comprising isolating or purifying the product.  
     
     
         23 . The method according to  claim 22  further comprising formulating the product into a composition comprising at least one additional component.