title: [Expression and purification of human sucrase protein in E.coli].
abstract: OBJECTIVE
  To construct prokaryotic expression vector pET-28a(+)-human sucrase (hSUC) and express hSUC fusion protein in E.coli.
  
  
  METHODS
  The hSUC gene fragment was amplified by reverse transciption PCR (RT-PCR) and cloned into pET-28a(+) vector to construct the prokaryotic expression vector pET-28a(+)-hSUC. The recombinant plasmid was then transformed into E.coli BL21. Hisdidine (His)-tagged fusion proteins were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nitrilotriacetic acid (Ni-NTA) agarose resin. The purified fusion proteins were identified by SDS-PAGE and Western blotting.
  
  
  RESULTS
  RT-PCR showed that sub-clone of hSUC was about 1482 bp. The recombinant plasmid was correctly constructed as demonstrated by sequencing and restriction enzyme analysis. The molecular mass of the fusion protein was about 61 240. Western blotting showed that the fusion proteins bound specifically to hSUC antibody.
  
  
  CONCLUSIONS
  The hSUC protein has been successfully expressed and purified in E.coli.
authors.affiliation: Department of Bioengineering, Zhuhai Campus, Zunyi Medical College, Zhuhai 519041, China.
authors.forename: Min
authors.initials: M
authors.lastname: Shao
authors.affiliation: Department of Bioengineering, Zhuhai Campus, Zunyi Medical College, Zhuhai 519041, China.
authors.forename: Xinying
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, Zhuhai Campus, Zunyi Medical College, Zhuhai 519041, China.
authors.forename: Qingfeng
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Bioengineering, Zhuhai Campus, Zunyi Medical College, Zhuhai 519041, China.
authors.forename: Xianli
authors.initials: X
authors.lastname: Niu
authors.affiliation: Department of Bioengineering, Zhuhai Campus, Zunyi Medical College, Zhuhai 519041, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, Zhuhai Campus, Zunyi Medical College, Zhuhai 519041, China. *Corresponding author, E-mail: hfztim@163.com.
authors.forename: Hefeng
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Department of Biochemistry, Zunyi Medical College, Zunyi 563003, China.
authors.forename: Zhenglong
authors.initials: Z
authors.lastname: Ge
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013393:Sucrase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D013393:Sucrase