title: [Fusion expression and affinity purification of a human novel gene tsbp in eukaryotic cells].
abstract: OBJECTIVE
  To construct an eukaryotic expressing vector of the human novel gene testis sperm binding protein (tsbp), and to express fusion protein and purify the recombinant protein by affinity chromatography.
  
  
  METHODS
  The novel gene tsbp was amplified by PCR from the prokaryotic expressing plasmid pGEX-5X-1/tsbp and an eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed after DNA recombination. After transfecting HEK293 cells with this recombinant vector via liposome mediation, the expression of the fusion protein was detected by RT-PCR, immunofluorescence and Western blot. Fusion protein His6-tsbp was purified from the cell lysis by immobilized metal affinity chromatography (IMAC) and the efficiency of purification was detected by SDS-PAGE and Western blot.
  
  
  RESULTS
  DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plasmid being transfected into HEK293 cells, RT-PCR verified the expression of tsbp mRNA. The result of immunofluorescence assay was positive and the fusion protein could be detected by Western blot of transfected HEK293 cells. The purified fusion protein could also be detected by SDS-PAGE and Western blot.
  
  
  CONCLUSIONS
  The novel gene tsbp was successfully cloned, expressed and purified in the form of His6 fusion protein, which is helpful for further study of the function of this testis sperm binding protein.
authors.affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China. suwenhui616@hotmail.com
authors.forename: Wen-hui
authors.initials: WH
authors.lastname: Su
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Li-yan
authors.initials: LY
authors.lastname: Zhang
authors.forename: Zhi-hong
authors.initials: ZH
authors.lastname: Zong
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.forename: Bing-zhi
authors.initials: BZ
authors.lastname: Yu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D029589:Seminal Plasma Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D013737:Testis / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; C425042:spermadhesin