Heterologous Production of Streptokinase in Secretory Form in Streptomyceslividans and in Nonsecretory Form in Escherichia coli

摘要

The skc gene encoding streptokinase (SK) with a molecularmass of approximately 47.4 kDa was cloned fromStreptococcus equisimilis ATCC 9542 and heterologouslyoverexpressed in Streptomyces lividans TK24 and E. coilusing various strong promoters. When the promoter forsprT [Streptomyces griseus trypsin (SGT)] was used in thehost S. lividans TK24, a 47.4-kDa protein was detected alongwith a smaller hydrolyzed protein (44 kDa), suggestingthat posttranslational hydrolysis had occurred as has beenreported in other expression systems. The casein/plasminogenplate assay revealed that the plasmid construct containingthe SGT signal peptide was superior to that containing theSK signal peptide in terms of SK production. Maximalproduction of SK was calculated to be about 0.25 unit/mlof culture broth, a value that was five times higher thanthat obtained with other expression systems using ermEand tipA promoters in the same host. When the skc genewas expressed in E. coli BL21(ΔDE3)pLys under thecontrol of the T7 promoter, a relatively large amount ofSK was expressed in soluble form without hydrolysis. SKactivity in E. coli/pET28a-T7pSK_m was more than 2 units/mlof culture broth, even though about half of the expressedprotein formed an inactive inclusion body.