An optimized protocol for overproduction of recombinant protein expression in Escherichia coli.
The gram-negative bacterium Escherichia coli (E. coli) offers a means for rapid, high-yield, and economical production of recombinant proteins. Here, a protocol for optimization of parameters involved in bacterial expression conditions is described. L-Asparaginase (ASNase II) was chosen as a model protein for our experiments. ASNase II gene (ansB) was cloned into the pAED4 plasmid and transformed into E. coli BL21pLysS (DE3)-competent cells. It was assumed that high cell density and high copy number of recombinant plasmid in the bacteria host could result in very high production of the recombinant protein. Circumstances for the overproduction of recombinant ASNase II including cell growth conditions, isopropyl β-D-1-thiogalactopyranoside (IPTG) level, ampicillin (Amp) concentration before and during IPTG induction, and cell density were optimized. Regarding the final optimization, overexpression of ASNase II was assessed on a large scale in LB medium. Periplasmic ASNase II was extracted using an alkaline lysis method. The extracted protein was purified by one-step DEAE-Sepharose fast-flow chromatography. ASNase II activity was considered an index for the protein expression. Applying the optimized practical protocol, protein production was significantly enhanced in comparison to the traditional IPTG induction method in the absence of a fermentor and can be applied for overexpression of other recombinant proteins.
a Research Center for Molecular Medicine , Hamadan University of Medical Sciences , Hamadan , Iran.
Elham
E
Bahreini
Khosrow
K
Aghaiypour
Roghayeh
R
Abbasalipourkabir
Mohammad Taghi
MT
Goodarzi
Massoud
M
Saidijam
Sedigheh Sadat
SS
Safavieh
D001215:Asparaginase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
D003470:Culture Media; D011994:Recombinant Proteins; D001215:Asparaginase