Expression and purification of human amelogenin in Escherichia coli / 华西口腔医学杂志
West China Journal of Stomatology
;
(6): 27-30, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-296724
ABSTRACT
<p><b>OBJECTIVE</b>To establish the expression and purification route for the gene encoding human amelongenin (AMG) mature peptide in Escherichia coli (E. coli).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>pGEX-4T-1/AMG system is used successfully to express human AMG fusing protein.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Escherichia coli
/
Amelogenin
/
Genetic Vectors
Limits:
Humans
Language:
Chinese
Journal:
West China Journal of Stomatology
Year:
2008
Type:
Article
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