High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions.
The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21 (DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 degrees C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg2+ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.
Yang
Qing
Q
Bioengineering Institute, Dalian University of Technology, Dalian 116012, P.R. China. yang_qing_dut@163.com
Xu
Jianqiang
J
Li
Min
M
Lei
Xuyu
X
An
Lijia
L
D000818:Animals; D002478:Cells, Cultured; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007477:Ions; D008670:Metals / Q000494:pharmacology*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D013696:Temperature; D013917:Thrombin / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D014757:Viper Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D017819:Viperidae / Q000378:metabolism
D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D014757:Viper Venoms; D013917:Thrombin; C557086:gloshedobin protein, Gloydius shedaoensis