[Expression and purification of human amelogenin in Escherichia coli].
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.
Zhang
Xue-yang
XY
Center of Implantology, Cuangdong Provincial Stomotological Hospital, Gangzhou 510280, China.
Zhao
Hua
H
Zhao
Hong-yu
HY
Wang
Chun-xian
CX
Zhang
Jin-cai
JC
D053523:Amelogenin; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans
D053523:Amelogenin