Patent ID: 8802394
Filing Date: 2014-08-12
Classification: C07K,C12N,C12P,C12Y

Abstract:
1. A method of expressing non-procaryotic biologically active disulfide-rich protein in prokaryotic host cells said method comprising the steps: a) obtaining a prokaryotic host cell transformed with an expression vector encoding a fusion protein under inducible control, said fusion protein comprising an N-terminal segment encoding thioredoxin and a C-terminal segment encoding said disulfide rich protein, wherein said host also carries stable mutations in thioredoxin reductase B (trxB) gene and/or the glutathione reductase (gor) gene, wherein said expression vector has an antibiotic resistance gene which makes it selectable on a first antibiotic, and wherein said trxB and gor mutations are selectable on at least one additional antibiotic to maintain the expression vector and trxB and gor mutations in said host cells during growth; b) growing the host cells of step a) in the presence of the first and said at least one additional antibiotic to obtain a sufficient number of cells suitable to seed a reactor in which host cells will be grown and the fusion protein expression induced; and c) seeding the reactor with the cells of step b) and growing the cells and inducing expression of the fusion protein, wherein said cells in the reactor are grown in the presence of the first antibiotic and in the absence of said at least one additional antibiotic.