Expression optimization and characterization of Tenebrio molitor antimicrobiol peptides TmAMP1m in Escherichia coli.
- Author:
Reyihanguli ALIMU
1
;
Xinfang MAO
;
Zhongyuan LIU
Author Information
1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, Xinjiang, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Anti-Infective Agents;
pharmacology;
Antimicrobial Cationic Peptides;
biosynthesis;
genetics;
pharmacology;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Insect Proteins;
biosynthesis;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
pharmacology;
Tenebrio;
chemistry
- From:
Chinese Journal of Biotechnology
2013;29(6):836-847
- CountryChina
- Language:Chinese
-
Abstract:
To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.
