Cloning, expression and purification of the outer membrane protein N from Gram-negative bacterial strains

Abstract

To employ polyclonal antibodies against 6his-tags to detect the expression of recombinant EcOmpN in the E. coli BL21 (DE3) Omp8 Rosetta strain using a Western blot, the ompN gene segments from four Gram-negative bacterial strains (including E. coli of BL21 (DE3) Omp8 Rosetta, DH5α, Origami, and XL1-blue) were combined with the pET21b(+) vector to create recombinant plasmids, known as pET21b(+)/ompN-BOR, pET21b(+)/ompN-DHA, pET21b(+)/ompN-Ori, and pET21b(+)/ompN-XLB for production of four recombinant EcOmpN proteins, called BL21 (DE3) Omp8 Rosetta (BOR), DH5α (DHA), Origami (Ori) and XL1-blue (XLB), respectively. Each recombinant plasmid-carrying bacterial strain was induced with 0.5 mM IPTG for 4 h. Crude recombinant EcOmpN proteins were separated on a 12% SDS-PAGE and transferred to a nitrocellulose membrane and treated separately with anti-OmpN and anti-histidine tag polyclonal antibodies. E. coli BL21 (DE3) Omp8 Rosetta strains fully produced all four recombinant EcOmpN proteins as inclusion bodies. Following protein refolding procedures, the secondary structure of recombinant BOR protein was likewise shown in the stable trimeric form, whereas recombinant DHA, XLB and Ori proteins were demonstrated in monomeric form. The intrinsic fluorescence and substance interaction of recombinant BOR protein were further established and showed that this protein reacted with kanamycin more intensely than ampicillin.