Escherichia coli is one of the most widely used host cells in foreign protein production through excessive expression for its fast growth rate, high concentration fungi cultivation and its economic production cost. Most widely used E. coli BL21(DE3) has a lower proteinase activity than general colon bacillus, and contains T7 polymerase gene for T7 promoter expression that allows an excessive expression of foreign proteins.
In E. coli BL21 (DE3), when rendering the expressed proteins in Ni-NTA column using imidazole, other target proteins as well as contaminated proteins are rendered and such contaminated proteins are not easily removed at a high concentration of imidazole. To solve this problem, two well known major contaminating proteins of E. coli BL21(DE3), Crp (catabolite gene activator) gene and SlyD (FKBP-type peptidyl-prolyl cis-trans isomerase) gene are selected, and its degree of contaminated protein reduction was measured using antisense RNA expression.
Based on the gene sequence of BL21(DE3)'s Crp and SlyD, Crp and SlyD genes are cloned to pET21c vector respectively, and they were excessively expressed to determine the gene expression. An expressive vector, pRSF Duet-1 vector, which has Kanamycin antibiotic resistant marker and therefore, is able to co-express with the target protein containing ampicillin antibiotic resistant marker, was inserted to Antisense RNA genes (As_crp, As_slyD, As_crp/As_slyD) respectively, and the antisense gene sequence was coded through DNA sequencing. By evaluating the gene expression hinderance through BL21(DE3) antisense gene expression, the reduction of contaminated proteins compared to the control group was not observed, but it affected the expression vector induced protein expression. Meanwhile, the contaminated protein expression hindrance through antisense method requires additional examinations such as western blot and RNA level reduction; it is expected that in Ni-NTA purification, the optimization of purification method in contaminated protein removal is more effective than host engineering.