Nucleic acids encoding recombinant protein A
View Patent ↗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 polypeptide consisting of SEQ ID NO:5.
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 sequence encoding the polypeptide is operably linked to an expression control sequence.
4. The isolated nucleic acid molecule of claim 1 , wherein the sequence encoding the polypeptide is operably linked to a bacterial ribosome binding site.
5. The isolated nucleic acid molecule of claim 4 , wherein the ribosome binding site is
ACGCGTGGAGGATGATTAA
(SEQ ID NO:3).
6. An expression vector comprising the nucleic acid molecule of claim 1 operably linked to an expression control sequence.
7. A bacterial cell comprising the vector of claim 6 .
8. A bacterial cell comprising the nucleic acid of claim 1 operably linked to an expression control sequence.
9. A bacterial cell transformed with the vector of claim 6 , or a progeny of the cell, wherein the cell expresses a truncated protein A polypeptide consisting of SEQ ID NO:5.
10. The bacterial cell of claim 7 , wherein the cell is a non-pathogenic bacterial cell.
11. The bacterial cell of claim 7 , wherein the cell is an Escherichia coli cell.
12. An Escherichia coli cell comprising an exogenous nucleic acid molecule that encodes a polypeptide consisting of SEQ ID NO:5.
13. An Escherichia coli cell comprising an exogenous nucleic acid molecule that encodes a polypeptide consisting of SEQ ID NO:7.
14. The cell of claim 13 , wherein the coding sequence is codon-optimized for expression in Escherichia coli.
15. A method of producing a truncated protein A polypeptide consisting of SEQ ID NO:5, the method comprising culturing the cell of claim 7 under conditions permitting expression of the polypeptide.
16. The method of claim 15 , further comprising purifying the truncated protein A polypeptide from the cytoplasm of the cell.
17. A method of producing affinity chromatography resin comprising a truncated protein A polypeptide consisting of SEQ ID NO:5, said method comprising performing the method of claim 16 and immobilizing the truncated protein A polypeptide on a solid support material.
18. The method of claim 17 , wherein the solid support material is selected from the group consisting of cellulose, agarose, nylon, and silica.
19. The method of claim 17 wherein the solid support material is a porous bead or a coated particle.
20. The method of claim 17 , wherein the solid support material is a controlled pore glass.
21. A method of purifying a protein comprising an Fc region of an IgG immunoglobulin, the method comprising
contacting the affinity chromatography resin comprising a polypeptide consisting of SEQ ID NO:5 made according to claim 17 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.