[Fusion expression and affinity purification of a human novel gene tsbp in eukaryotic cells].
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.
Su
Wen-hui
WH
Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China. suwenhui616@hotmail.com
Zhang
Jie
J
Zhang
Li-yan
LY
Zong
Zhi-hong
ZH
Zhang
Zhe
Z
Yu
Bing-zhi
BZ
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*
D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; C425042:spermadhesin