Construction Of A Modified Escherichia Coli BL21(DE3)

This study used Red recombination system to modify Escherichia coli BL21(DE3),a popular protein production strain. A mutant E.coli BL21(DE3) with inactivated lpxM and a modified E.coli protein production strain with capable of autohudrolysing host nucleic acid were constructed in order to reduce LPS contamination and nucleic acid contamination in the expressed protein and decrease the viscosity of cell lysates which is caused by high molecular weight chromosomal DNA of the host cell.1 Mutations in the lpxM gene produce a low endotoxicity lipopolysaccharide. Having constructed a targeting vector with about 500bp long arms homologous to lpxM, kanamycin resistance gene was inserted in between the two regions. The replacement fragment was electroporated into a recipient strain expressing the highly proficient homologous recombinant system encoded by plasmid pKOBEG After target gene was inserted by the kanamycin gene,the resistance gene was then eliminated by using pCP20 encoding the FLP recombinase. Two PCR procedures was used to confirm the lpxM gene was inactivated by insertion and SDS-PAGE was applied to analyse LPS and protein expression. According to electrophosis pattern, LPS extracted from the mutant BL21 strain was different from one from the original strain.The mutant E.coli BL21(DE3) had the same capacity to support the production of recombinant proteins as the original strain. This study provides a viable knock-in site in E.coli BL21 (DE3) and The mutant E.coli BL21(DE3) strain with inactivated lpxM gene could be useful to produce recombinant proteins with reducing the risk linked with low level of endotoxin contamination.2 High molecular weight chromosomal DNA is responsible both for the viscosity of cell lysates,and it is a source of heterologous DNA sequences whose inclusion in the final product must be prevented. To resolve the problem which is caused by the host nucleic acid, We have constructed a modified Escherichia...