Cloning and expression of an actinokinase gene from a thermophilic Streptomyces in Escherechia coli.
A thermophilic Streptomyces megasporus strain SD5, could secrete a new fibrinolytic (actinokinase) at 55 degrees C. The gene (ackS) encoding actinokinase was isolated from the chromosomal DNA of S. megasporus SD5 and cloned in different hosts and vectors. The expression was obtained in E. coli JM109 using Cla I linearized pBR322 as vector (pSR 500). The recombinant E. coli containing pSR 500 expressed active actinokinase but the expression was low and the recombinant was unstable in liquid culture. Deletion analysis revealed that removal of Bam H I-Sal I fragment from down stream and Cla I-EcoRI from upsream enhanced the stability and expression of ackS in both solid and liquid media. For over expresion, the ackS gene was cloned in E. coli C 600 using Bam HI linearized pT7-7. This seemed to be the most suitable host vector system. The recombinant and native form of actinokinase exhibited similar characteristics. Actinokinase was the first thrombolytic enzyme from a thermophile to be cloned and over expressed in a mesophilic heterologous expression system.
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Division of Microbiology, MACS-Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India.
Dey
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D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005342:Fibrinolysis; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D013300:Streptokinase / Q000235:genetics*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D013300:Streptokinase; C437863:actinokinase