Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli.
Previously, we constructed human interleukin-6 (hIL-6)-secreting Escherichia coli and Salmonella typhimurium strains by fusion of the hIL-6 cDNA to the HlyA(s) secretional signal, utilizing the hemolysin export apparatus for extracellular delivery of a bioactive hIL-6-hemolysin (hIL-6-HlyA(s)) fusion protein. Molecular analysis of the secretion process revealed that low secretion levels were due to inefficient gene expression. To adapt the codon usage in hIL-6 cDNA to the E. coli codon bias, a synthetic hIL-6Ec gene variant was constructed from 20 overlapping oligonucleotides, yielding a 561-bp fragment, which comprises the complete hIL-6 cDNA sequence. Genetic fusion of the hIL-6Ec gene with the hlyA(s) secretional signal as an integral part of the hemolysin operon resulted in 3-fold higher hIL-6-HlyA(s) secretion levels in E. coli, compared to a strain expressing the original hIL-6-hlyA(s) fusion gene. An increase in the electrophoretic mobility of secreted hIL-6-HlyA(s) in non-reducing SDS-PAGE, similar to that found for recombinant mature hIL-6, and the absence of such a mobility shift in the intracellular hIL-6-HlyA(s) protein fraction indicated that in hIL-6-HlyA(s) most probably correct intramolecular disulfide bond formation occurred during the secretion step. To confirm the disulfide bond formation, hIL-6-HlyA(s) was purified by a single-step immunoaffinity chromatography from culture supernatant in yields of 18 microg/L culture supernatant with purity in the range of 60%. These results demonstrate that codon usage has an impact on the hemolysin-mediated secretion of hIL-6 and, furthermore, provide evidence that the hemolysin system enables secretory delivery of disulfide-bridged proteins.
Chirurgische Forschung, Chirurgische Universitätsklinik, Hugstetter Strasse 55, D-79106 Freiburg i. Br., Germany
Yuanyi
Y
Li
Carol X
CX
Chen
Bernd-Ulrich
BU
von Specht
Heinz P
HP
Hahn
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D003062:Codon; D004220:Disulfides; D006460:Hemolysin Proteins; D015850:Interleukin-6; D011994:Recombinant Proteins