METHODS OF PRODUCING TWO CHAIN PROTEINS IN BACTERIA

摘要

Provided herein are methods of producing a polypeptide containing two chains, such as an antibody including a light chain and a heavy chain. In particular, methods are provided for producing heterologous secretory proteins in bacteria through utilization of optimized expression vectors and culture processes.

主权项

1. A method of producing a polypeptide comprising two chains in a prokaryotic host cell, the method comprising:
(a) culturing the host cell to express the two chains of the polypeptide in a culture medium under conditions comprising:a growth phase comprising a growth temperature and a growth agitation rate, anda production phase comprising a production temperature and a production agitation rate,whereby upon expression the two chains fold and assemble to form a biologically active polypeptide in the host cell;
wherein the host cell comprises a polynucleotide comprising(1) a first translational unit encoding a first chain of the polypeptide;(2) a second translational unit encoding a second chain of the polypeptide; and(3) a third translational unit encoding at least one chaperone protein selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate; and (b) recovering the biologically active polypeptide from the host cell.