title: [Cloning and expression of Hgp44 from Porphyromonas gingivalis].
abstract: OBJECTIVE
  To clone and express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis (Pg) and to purify the protein.
  
  
  METHODS
  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.
  
  
  RESULTS
  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 data showed that the protein was 44 000 in size and expressed mostly in the form of inclusion body. The purification of fusion protein was achieved using Ni(2+) affinity chromatography. About 3.5 mg/L fusion protein was obtained.
  
  
  CONCLUSIONS
  Hgp44 was successfully expressed in the prokaryotic expression system and purified by IMAC using a Ni(2+) matrix column.
authors.affiliation: Department of Operative Dentistry and Endodontics, Hospital of Stomatology, College of Medicine, Zhejiang University, Hangzhou 310006, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Hong-hua
authors.initials: HH
authors.lastname: Cui
authors.forename: Qi-Xia
authors.initials: QX
authors.lastname: Zhang
authors.forename: Shu-guang
authors.initials: SG
authors.lastname: Han
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002999:Clone Cells; D003001:Cloning, Molecular; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins