title: [Expression and purification of human amelogenin in Escherichia coli].
abstract: OBJECTIVE
  To establish the expression and purification route for the gene encoding human amelongenin (AMG) mature peptide in Escherichia coli (E. coli).
  
  
  METHODS
  Recombined plasmid pGEX-4T-1/AMG was identified by double endonuclease digestion electrophoretogram and DNA sequence analysis. The recombined plasmid was transformed to E. coli BL21. The inducing time, isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration and inducing temperature were optimized for the express system. Under the optimized condition, the target fusing protein in superatant, periplasm, plasm and inclusion body was analyzed separately. A great amount of target fusing protein was found in the dissoluble protein. AMG fusing protein was purified by the GSTrapFF affinity column.
  
  
  RESULTS
  Double endonuclease digestion electrophoretogram and DNA sequence analysis were done to identify the recombined vector pGEX-4T-1/AMG. The results were consistent with the anticipation. The optimum inducing time was 14.5 hours. The optimum IPTG concentration was 1.0 mmol/L. The optimum inducing temperature was 20 degrees C. Under this condition, the target protein was expressed to a maximum. Plentiful target protein was expressed in plasm and inclusion body under the optimized condition. A mount of plasm protein was obtained and purified by the GSTrapFF affinity column. The purified liquid was collected and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PACE). The protein electrophoresis map showed that AMG fusing protein was purified successfully. After twice elution, high pure fusing protein was obtained.
  
  
  CONCLUSIONS
  pGEX-4T-1/AMG system is used successfully to express human AMG fusing protein.
authors: Center of Implantology, Cuangdong Provincial Stomotological Hospital, Gangzhou 510280, China.
authors: Xue-yang
authors: XY
authors: Zhang
authors: Hua
authors: H
authors: Zhao
authors: Hong-yu
authors: HY
authors: Zhao
authors: Chun-xian
authors: CX
authors: Wang
authors: Jin-cai
authors: JC
authors: Zhang
mesh_terms: D053523:Amelogenin; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans
chemical_list: D053523:Amelogenin