关键词: 人釉原蛋白, 融合蛋白, 纯化

Abstract:

Objective To establish the expression and purification route for the gene encoding human amelogenin（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 supernatant,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 ℃. 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（SDSPAGE）. The protein electrophoresis map showed that AMG fusing protein was purified successfully. After twice elution, high pure fusing protein was obtained. Conclusion pGEX- 4T- 1/AMG system is used successfully to express human AMG fusing protein.

Key words: human amelogenin, fusing protein, purification