title: [Prokaryotic expression and purification of human TLE1 N-terminal Q domain fragment and production of its polyclonal antibody].
abstract: BACKGROUND
  TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody.
  
  
  METHODS
  The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST). Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136) fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136) for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot.
  
  
  RESULTS
  The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14,000 Da) is the interest protein TLE1-Q(1-136). The TLE1 N-terminal Q domain fragment TLE1-Q(1-136) and its polyclonal antibody have been acquired, with an antibody titer of 1:20,000.
  
  
  CONCLUSIONS
  Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136) and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.
authors.affiliation: Department of Thoracic Surgery, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Su
authors.initials: S
authors.lastname: Wang
authors.forename: Zhifei
authors.initials: Z
authors.lastname: Xu
authors.forename: Hua
authors.initials: H
authors.lastname: Tang
authors.forename: Lingyun
authors.initials: L
authors.lastname: Wei
authors.forename: Xuewei
authors.initials: X
authors.lastname: Zhao
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D056970:Co-Repressor Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007114:Immunization; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D056970:Co-Repressor Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C499043:TLE1 protein, human