Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli

Abstract

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 μg/ 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.