Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

被引:10
作者
Avilan, L [1 ]
Yarzabal, A [1 ]
Jurgensen, C [1 ]
Bastidas, M [1 ]
Cruz, J [1 ]
Puig, J [1 ]
机构
[1] Univ Los Andes, Fac Ciencias, LABIOMEX, Lab Biol & Med Expt, Merida 5101, Venezuela
关键词
recombinant streptokinase; plasminogen activators; protein purification;
D O I
10.1590/S0100-879X1997001200007
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a K-m = 0.70 mu M and k(cat) = 0.82 s(-1), when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.
引用
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页码:1427 / 1430
页数:4
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