Heterologous production of streptokinase as a secretory form in Streptomyces lividans and nonsecretory form in Escherichia coli.
The skc gene encoding streptokinase (SK), with a molecular weight of approximately 47.4 kDa, was cloned from Streptococcus eouisimilis ATCC9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the sprT promoter was used in the S. lividans TK24 host, the SK protein corresponding to 47.4 kDa was detected with a smaller hydrolyzed protein (44 kDa), implying posttranslational hydrolysis occurred as reported in other expression systems. Casein/plasminogen plate assay revealed that plasmid construct with the signal peptide of SK was superior to that with the signal peptide of sprT in SK production. The maximum productivity of SK was calculated as less than 0.25 unit/ml of the culture broth, which was similar level to those from other expression systems hiring ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(DE3)pLys under control of T7 promoter, relatively large amount of SK was expressed in soluble form without hydrolyzed protein. The SK activity produced by E. coli/pET28a-T7pSKm was more than 2 units/ml of culture even though about half of the expressed protein formed inactive inclusion body.
Department of Biological Science, Myongji University, Yongin 449-728, Korea.
Mi-Ran
MR
Kim
Yong-Hoon
YH
Choeng
Won-Jae
WJ
Chi
Dae-Kyung
DK
Kang
Soon Kwang
SK
Hong
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008970:Molecular Weight; D021382:Protein Sorting Signals; D021381:Protein Transport; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048170:Streptomyces lividans / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D013300:Streptokinase