PMID: 22333354Feb 16, 2012Paper

Cloning and expression of Hgp44 from Porphyromonas gingivalis

Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology
Jie ZhangHui Chen

Abstract

To clone and express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis (Pg) and to purify the protein. The genomic DNA of Pg was isolated from PgATCC33277. The Hgp44 gene fragment was amplified by polymerase chain reaction (PCR) and then inserted into the cloning vector pMD18-T and sequenced. The correct fragment was linked with a prokaryotic expression vector pET-22b to construct the recombinant expression plasmid pET22b-Hgp44. The pET22b-Hgp44 confirmed by enzyme digestion was transformed into competent Escherchia coli (Ec) BL21 (DE3) cells. Expression of fusion protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and purified by immobilized metal-chelating affinity chromatography (IMAC) using a Ni(2+) matrix column. SDS-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis were used to examine the fusion protein. A 1 100 bp fragment was successfully amplified and verified by the agarose gel electrophoresis and sequencing. The generated recombinant expression vectors pET22b-Hgp44 were verified by enzyme digestion and agarose gel electrophoresis. The expression of fusion protein in Ec BL21 (DE3) cells was examined by SDS-PAGE and Western blotting analyses, and the da...Continue Reading

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