Study on the optimal production and purification of antibacterial peptide mastoparan with various gene constructions

• Main Authors: Sung-Huan Chung, 鐘崧桓
• Other Authors: Yung-Chuan Liu
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/40542999058628785934

碩士 === 國立中興大學 === 化學工程學系所 === 105 === Mastoparan is an antimicrobial peptide extracted from the Vespa, which has the effect of killing Gram-positive bacteria, Gram-negative bacteria and fungi, and has very low damage to eukaryotic cells. It was of great potential for production of antimicrobial drugs. However, if directly extracted from the biological, the production amount will be small and expensive. The application of the gene transfer to the recombinant strain will be able to reduce the production costs. In this study, we use 4 kinds of mastoparan, i.e., MP-AF, MP-B, MP-D and MP-M, which were obtained from the venom of Vespa species in Taiwan. The fusion protein was constructed with its N-terminal designed with chitin-binding-domain plus ssp dnaB intein (CBD-INT) and and C-terminal with 6 histidines. In this way, the affinity adsorption columns such as CBD affinity tag column and poly his-tag column can be used for the mastoparans purification. Owing to the toxicity of mastoparans, the construction of plasmid is extremely unstable. The direct fusion protein expression is very low. Many different induction conditions were tried to optimize the soluble protein production. It was found that when yeast extract was added during the IPTG induction in the late logarithmic phase, the soluble protein yield could be enhanced more than 20 times, the target protein before purification (CBD- INT-MP-M-6His) is about 200 mg / L. The large amount of soluble proteins would facilitate the subsequent purification studies.