Use of high density cultures of Escherichia coli for high level production of recombinant Pseudomonas aeruginosa exotoxin A.
An efficient fermentation method for the production of two modified recombinant Pseudomonas aeruginosa exotoxin As cloned in Escherichia coli BL21(lambda DE3) was developed. Cell densities of 16-30 g dry weight/1 were found to be most suitable for the induction of protein synthesis, which was under the isopropyl beta-D-thiogalactopyranoside (IPTG)-inducible T7 expression system. A concentration of 0.6 mM IPTG and induction time of 90 min were found to give the best results for production of the modified toxins. Using this procedure, gram amounts of the proteins were obtained in a 3-1 bench-top fermentor. The high density growth of the bacteria did not impair the integrity of the proteins and did not interfere with the purification procedure.
Fass
R
R
Biotechnology Unit, LCDB, NIDDK, NIH, Bethesda, MD 20892.
van de Walle
M
M
Shiloach
A
A
Joslyn
A
A
Kaufman
J
J
Shiloach
J
J
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D001431:Bacteriological Techniques; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005098:Exotoxins / Q000096:biosynthesis* / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D007544:Isopropyl Thiogalactoside; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A