毕赤酵母中人Ⅲ型胶原蛋白α链与病毒羟脯氨酸酶的共表达  

Recombinant coexpression for hydroxylated human type Ⅲ collagen alpha chain with a viral prolyl 4-hydroxylase in Pichia pastoris

作　　者：史静静[1] 高源[1] 贺婧[1] 马晓轩[1] 

机构地区：[1]西北大学化工学院,陕西西安710069

出　　处：《西北大学学报（自然科学版）》2017年第2期231-236,共6页Journal of Northwest University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(21576223)

摘　　要：将人Ⅲ型胶原α链与一种来源于病毒的4-羟脯氨酸酶在毕赤酵母中共表达,以获得能满足应用需求的羟基化胶原蛋白。分别构建包含酶与胶原基因的重组质粒p PICZBL593和pPIC9K-COL3A1,并将质粒均转入毕赤酵母GS115中以表达这两种蛋白。质粒测序结果表明目的基因成功插入到载体中;实时定量PCR结果显示两种基因均在mRNA水平表达;SDS-PAGE和Western Blot进一步证实两种蛋白在毕赤酵母GS115中的成功表达;氨基酸组成分析和质谱结果证实表达的胶原中含有羟脯氨酸。获得羟基化的胶原蛋白。In order to obtain hydroxylated collagen （Hyp-containing collagen） for satisfying the application, human collagen αl （ Ⅲ ） chain in Pichia pastoris was co-expressed with a viral prolyl 4-hydroxylase L593 in this study. The two recombinant plasmids pPICZB-L593 and pPIC9K-COL3A1 were constructed and trans- formed into GS115 for expressing proteins. The sequencing results showed that the target genes were inserted into recombinant vectors successfully. Real-Time quantitative PCR indicated that they both expressed on mR- NA level. SDS-PAGE and Western Blot results further proved the successful expression of target proteins by GSll5. Hydroxyproline of collagen was confirmed by amino acid composition analysis and LC-MS/MS spec- tra. This research opens up a new possibility and provides a reference for production of hydroxylated collagen in Pichia pastoris.

关 键 词：共表达 4-羟脯氨酸酶 人Ⅲ型胶原α链 羟基化 

分 类 号：Q3[生物学—遗传学]