摘要
为了寻找毕赤酵母表达虎纹捕鸟蛛毒素-Ⅰ(HWTX-Ⅰ)时产生不均一性表达产物的原因,应用阳离子交换层析、反相HPLC、Tricine SDS-PAGE电泳、质谱等技术,对基因工程菌Pichia pastorisGS115/HWTX-Ⅰ表达的不均一产物进行了分离、纯化和鉴定,并对不均一产物的N端和C端进行测序,结果表明表达产物的不均一性主要体现在重组HWTX-Ⅰ的N端信号肽加工的不完全以及C末端的内部降解。生物学活性分析表明该不均一产物的活性仅为天然HWTX-Ⅰ活性的30%。同时,对如何避免不均一性表达产物的产生提出了相应的对策。
To seek the reason of heterogeneity of recombinant HWTX-Ⅰ（rHWTX-Ⅰ ） expressed in Pichia pastoris. We expressed HWTX-Ⅰ gene of interest in Pichia pastoris GS115/HWTX-Ⅰ.The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-Ⅰresults from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-Ⅰ only showed 30% activity compared with the native HWTX-Ⅰ.The Solutions to how to avoid the heterogeneity are also discussed.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第2期215-219,共5页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目(No.39570392)资助。~~
