High-level expression of streptokinase gene in escherichia coli and development of aprE-lacUV5 hybrid promoters

Abstract

The expression vector for streptokinase has been constructed from our previously cloned streptokinase coding gene($\underline{\mbox{skc}}$). Because of its deleterious effect on the host cell growth, the leader sequence of skc was removed and the leader sequence-deleted $\underline{\mbox{skc}}$ was subcloned into the vector pkk223-3, which contains the regulatable $\underline{\mbox{tac}}$ promoter and $\underline{\mbox{rrnB}}$ $T_1T_2$ transcription terminator, with a short synthetic oligonucleotide adapter. When this vector, pKS601, was expressed in $\underline{\mbox{Escherichia}}$ $\underline{\mbox{coli}}$, a 47.4 kD protein was found to be newly accumulated to about 12% of the total cellular proteins and it was identified as the streptokinase by immunoblotting with rabbit anti-streptokinase polyclonal serum. The expressed leader sequence-deleted streptokinase caused no detrimental effect to host cell physiology, which means that the deleterious effect of the wild-type $\underline{\mbox{skc}}$ had caused by its putative leader sequence, and showed no degraded 44 kD streptokinase, which is characteristic feature of streptokinase expressed from wild-type $\underline{\mbox{skc}}$. At 37℃, one-fourth of streptokinase was found in sonication-insoluble pellet, which regarded as inclusion bodies, but at 25℃, nearly all of streptokinase was found in sonication-soluble fraction. But the total activity of expressed streptokinase showed the highest values when cells had cultured and induced at 37℃, The expressed streptokinase was purified to near homogeneity using DEAE-cellulose and Sephadex G-150 column. Its specific activity was 1.3×$10^5$ CLN units/mg protein. And a series of $\underline{\mbox{E.}}$ $\underline{\mbox{coli}}$-$\underline{\mbox{Bacillus}}$ $\underline{\mbox{subtilis}}$ shuttle vectors was constructed by fusing and manipulating the $\underline{\mbox{Staphylococcus}}$ $\underline{\mbox{aureus}}$ plasmid pUB110 and $\underline{\mbox{E.}}$ $\underline{\m...