Construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae and its expression in E. coli.
In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) efflux system, plasmid pET-28a(+) encoding mtrC gene was constructed and the related target protein was expressed in Escherichia coli (E. coli) DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with restriction endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuclease and DNA sequencing. Its sequence was 99.5% homologus to that published on GeneBank (U14993). A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.
Chen
Hongxiang
H
Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Tu
Yating
Y
Lin
Nengxing
N
Huang
Changzheng
C
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; C094589:MtrC protein, Neisseria gonorrhoeae; D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes