[The expression of gene related to salt tolerance from Sinorhizobium meliloti 042BM in Escherichia coli and purification of its fusion protein].
A 1.9kb DNA fragment related to salt tolerance of S. meliloti strain 042BM containing two open reading frames were obtained by PCR amplification and ligated into shuttle vector pBBR1-MCS2. The complementation experiment showed that ORF2 is related to salt tolerance and named as rstA gene. Then the gene was cloned into the expression vector pThio-HisA, B and C, respectively, and recombinant expression vectors pGSA, pGB and pGC were constructed, and transformed into E. coli Top10. Inducing by IPTG and analyzing with SDS-PAGE, the fusion protein encoded by pGSA was obtained,and it is 36% content of whole cell protein. It was isolated and purified by affinity chromography on ProBond, and the inclusion body precipitated by saturated sulfate ammonium, and 95% purity of fusion protein was obtained. The final product displayed a single band with a corresponding molecular weight 43kD in SDS-PAGE, and was verified by the Western blot.
Department of Microbiology, College of Biological Sciences, Key Laboratory for Agro-Microbial Resources and Application of Agriculture Ministry, China Agricultural University, Beijing 100094, China.
Shi-chao
SC
Ge
Lei
L
Wang
Xiao-hong
XH
Li
Su-wei
SW
Qi
Su-sheng
SS
Yang
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D020579:Sinorhizobium / Q000187:drug effects / Q000235:genetics*; D012965:Sodium Chloride / Q000494:pharmacology*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride