摘要
棉铃虫组织蛋白酶B(Helicoverpa armigeraCathepsin B,HCB)属于半胱氨酸蛋白酶类,参与胚胎发育中卵黄蛋白水解供给胚胎发育的氨基酸。将HCB基因克隆到pPIC9K载体并转化毕赤酵母KM71菌株,经甲醇诱导,HCB表达并分泌到培养上清中。表达产物经SDS-PAGE测定分子量为38kDa,与HCB基因编码的蛋白质分子量一致。用HCB的特异性抗体检测表明重组表达产物为棉铃虫组织蛋白酶B,原位水解实验显示重组表达的蛋白酶具有蛋白水解活性,表明在毕赤酵母中表达了有活性的棉铃虫组织蛋白酶B,可用于组织蛋白酶B酶原活化机理研究及开发新蛋白酶产品。
Cathepsin B from Helicoverpa armigera （HCB） belongs to the group of cysteine proteinases. HCB is proved being involved in the degradation of yolk proteins during embryonic development, which is an acidic preferring enzyme and is resistant to SDS. The expression of the proenzyme may offer a model for investigating the activation of the enzyme. The HCB gene was constructed into pPIC9K and expressed in Pichia pastoris KMTI strain . After induction by methanol, HCB was expressed and secreted into the medium. The molecular weight of the recombinant procathepsin B was determined as about 38 kDa. The expressed product was confirmed to be HCB by immunoblotting assay using specific rabbit anti-HCB polyclonal antibody. The activity of the product was assayed by in situ hydrolysis （gelatin-SDS-PAGE）. These results showed that HCB with proteolytic activity was expressed in P. pastoris KMT1. This proenzyme can be used for further research on the activation of the proenzyme or industrial production.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2006年第2期44-48,共5页
China Biotechnology
基金
国家自然科学基金资助项目(30330070)
关键词
棉铃虫组织蛋白酶B
毕赤酵母
重组表达
Helicoverpa armigera Cathepsin B Pichia pastoris Recombinant expression
