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標題: 不同基因構築之抗菌胜肽mastoparan 生產最適化與純化之研究
Study on the optimal production and purification of antibacterial peptide mastoparan with various gene constructions
作者: 鐘崧桓
Sung-Huan Chung
關鍵字: mastoparan;抗菌胜肽;生產最適化;質體不穩定性;多重抗藥性;mastoparan;antimicrobial peptides;optimal production;plastid instability;multiple drug resistance
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本研究利用基因工程的方法生產抗菌胜肽mastoparan,取用4種台灣常見虎頭蜂的mastoparan抗菌胜肽,MP-AF、MP-B、MP-D和MP-M,在蛋白上的N端和C端分別設計了chitin-binding-domain(CBD)和6個Histidine,期望以親和性吸附管柱CBD affinity tag column和poly his tag column來進行純化。然而mastoparan本身為毒蛋白,且實驗發現質體很不穩定,直接導致了蛋白表現量極低。因此我們採用了許多不同的誘導條件優化蛋白生產,探討出在誘導前加入營養物yeast extract並在生長對數期後期和15℃誘導,可以大大的提升可溶性蛋白產量20倍以上,純化前的目標蛋白(CBD-INT-MP-M-6His)的產量約為200mg/L,在大量的蛋白表現下,純化步驟會變得容易許多,以利於後續的研究。

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.
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