Nucleic acids encoding recombinant protein a

Abstract

Disclosed are new recombinant nucleic acids encoding protein A polypeptides and methods of using these nucleic acids.

1 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a truncated protein A polypeptide that
 (i) includes some portion of, but less than all of, a complete native X domain;   (ii) lacks a signal sequence; and   (iii) binds specifically to an Fc region of an IgG immunoglobulin.   
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the coding sequence is codon-optimized for expression in a non-pathogenic organism. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid comprises a sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         4 . The isolated nucleic acid molecule of  claim 3 , wherein the nucleic acid comprises a sequence at least 95% identical to SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         5 . The isolated nucleic acid molecule of  claim 4 , wherein the nucleic acid comprises SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the sequence encoding a truncated protein A polypeptide is operably linked to a bacterial ribosome binding site. 
     
     
         7 . (canceled) 
     
     
         8 . An expression vector comprising the nucleic acid molecule of  claim 1  operably linked to an expression control sequence. 
     
     
         9 . A bacterial cell comprising the vector of  claim 8 . 
     
     
         10 . (canceled) 
     
     
         11 . A bacterial cell transformed with the vector of  claim 8 , or a progeny of the cell, wherein the cell expresses a truncated protein A polypeptide. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . A method of producing a truncated protein A polypeptide, the method comprising culturing the cell of  claim 9  under conditions permitting expression of the polypeptide. 
     
     
         15 . The method of  claim 14 , further comprising purifying the truncated protein A polypeptide from the cytoplasm of the cell. 
     
     
         16 . A method of producing a truncated protein A polypeptide-containing affinity chromatography resin, said method comprising performing the method of  claim 15  and immobilizing the truncated protein A polypeptide on a solid support material. 
     
     
         17 - 19 . (canceled) 
     
     
         20 . A method of purifying a protein comprising an Fc region of an IgG immunoglobulin, the method comprising
 contacting the protein A polypeptide-containing affinity chromatography resin made according to  claim 16  with a solution comprising a protein comprising an Fc region of an IgG immunoglobulin;   washing the substrate; and   eluting bound protein comprising an Fc region of an IgG immunoglobulin.   
     
     
         21 . An  E. coli  cell comprising an exogenous nucleic acid molecule that encodes a polypeptide consisting of SEQ ID NO:7. 
     
     
         22 . The cell of  claim 21 , wherein the coding sequence is codon-optimized for expression in  E. coli.    
     
     
         23 . An isolated nucleic acid molecule that encodes a polypeptide comprising one or more nucleic acid sequences encoding a  S. aureus  protein A Ig-binding domain and a portion of a  S. aureus  protein A X-domain, wherein the nucleic acid sequence encoding the portion of the X-domain has a stop codon at position 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, or 409 of the X domain coding sequence. 
     
     
         24 . The nucleic acid of  claim 23 , wherein the one or more sequences encoding an Ig binding domain are wild-type. 
     
     
         25 . The nucleic acid of  claim 23 , wherein the one or more sequence encoding an Ig binding domain are codon-optimized. 
     
     
         26 . The nucleic acid of  claim 23 , wherein the sequence encoding the X domain is wild-type except for the stop codon. 
     
     
         27 . The nucleic acid of  claim 23 , wherein the sequence encoding the X domain is codon-optimized. 
     
     
         28 . (canceled)