[Study of HSP70 gene upstream regulation element on expression efficiency of GST gene in M. smegmatis].
Four different expression vectors were constructed by cloning foreign gene which encode Schistosoma japonicum 26 kD antigen (Sj26GST) into Escherichia coli-Mycobacteria shuttle plasmid pBCG-2000 and investigated their expression efficiency in mycobacteria smegmatis. The plasmid which contains promoter of human mycobacterial tuberculosis heat shock protein 70 was firstly digested with Nco I and modified with two different ways to lead to two kinds of SD sequences, and ligated with Sj26GST encoding gene. Then, the DNA fragment contained HSP70 promoter and Sj26GST gene was obtained and cloned into E. coil-mycobacteria shuttle plasmid pBCG-2000, and finally four recombinant mycobacterial expression vectors that differenciated in SD sequence, orientation and copy number were selected. The expressed native recombinant Sj26GST(rSj26GST) was soluble and could be observed on SDS-PAGE about at the molecular weight of 26 kD obviously. Analysis with protein density scanning indicated: the expression efficiency that containing double-copy promoter-foreign gene vector was the highest and the expressed protein which was about 1.6 times than others was 28% of total protein of Mycobacteria smegmatis. The cloned direction and SD sequence had no significant effect on expression efficiency.
Cheng
J
J
Department of Medical Molecular Biology, Research Center of Experimental Medicine, Tongji Medical University, Wuhan 430030.
Huangfu
Y
Y
Hai
T
T
Liang
J
J
Xiao
H
H
D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D012549:Schistosoma japonicum / Q000276:immunology
D000947:Antigens, Helminth; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase