title: [Prokaryotic expression, purification and identification of  NY-ESO-1/GST fusion protein in E.coli].
abstract: OBJECTIVE
  To construct an expression plasmid for NY-ESO-1 gene and identify the expression of recombinant protein NY-ESO-1/GST in E.coli.
  
  
  METHODS
  NY-ESO-1 segment was amplified from the testis cDNA library by RT-PCR and cloned into the prokaryotic expression vector pGEX4T-1 downstream tagged by GST to construct the expression plasmid pGEX-4T1-NY-ESO-1. The recombinant vector was transformed to BL21 (DE3) and NY-ESO-1/GST fusion protein was induced expression by IPTG. The protein was purified by urea elution and identified by SDS-PAGE and Western blotting.
  
  
  RESULTS
  The NY-ESO-1 segment was successfully amplified and its sequence was identical with that published in GenBank. The BL21 (DE3) pLysS containing the pGEX-4T1-NY-ESO-1 expressed a M(r); 44 000 fusion protein under the induction of IPTG. The purity of the protein was 90%. Western blotting  proved  that NY-ESO-1/GST had a specific reaction with anti-GST mAb.
  
  
  CONCLUSIONS
  The prokaryotic expression vector of NY-ESO-1 has been constructed and the fusion protein NY-ESO-1/GST of high purity is successfully expressed.
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Tang
authors.forename: Chao-jun
authors.initials: CJ
authors.lastname: Song
authors.forename: Yuan-jie
authors.initials: YJ
authors.lastname: Sun
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Yu-ying
authors.initials: YY
authors.lastname: Wei
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
chemical_list: D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside