人壳三糖酶基因在巴氏毕赤酵母中的真核表达和纯化  

Expression and Purification of Chitotriosidase in Methylotrophic Yeast Pichia Pastoris

作　　者：刘斌[1] 沈柱[1] 陈凌[2] 刘玉峰[1] 菅金龙[3] 

机构地区：[1]第四军医大学西京医院皮肤科,陕西西安710032 [2]第三军医大学大坪医院皮肤科,重庆400042 [3]第四军医大学基础部免疫学教研室,陕西西安710032

出　　处：《中国皮肤性病学杂志》2007年第6期337-339,共3页The Chinese Journal of Dermatovenereology

摘　　要：目的用巴斯德毕赤酵母系统表达人壳三糖酶,并进行亲和层析纯化,获得具有天然构象的人壳三糖酶。方法从pUC19-Chit融合载体中扩增编码人壳三糖酶基因,并将其克隆入真核表达载体pPIC9K,以电穿孔法转化酵母GS115。用MD平板筛选重组子,G418筛选高拷贝转化子,经甲醇诱导表达后,几丁质亲和层析法对表达上清进行纯化,并进行SDS-PAGE分析。结果构建了人壳三糖酶融合真核表达载体pPIC9K-Chit。SDS-PAGE显示人壳三糖酶分子量约为50000Da,表达量约为24mg/L,纯度为97%。结论人壳三糖酶在毕赤酵母中的成功表达和具有天然构象的人壳三糖酶获得为进一步研究其抗真菌作用提供了物质基础。Objective To express and purify human Chitotriosidase in methylotrophic yeast Pichia pastoris and obtain Chitotriosidase protein with natural structure. Methods Gene encoding human Chitotriosidase, amplified by PCR from pUC19-Chit vector, was cloned into the vector pPIC9k. The constructed plasmid was transformed into yeast GS115 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of Chitotriosidase was induced by addition of methanol. The supernatant was collected and purified with chitin affinity column chromatography and analyzed by SDS-PAGE. Results The recombinant vector of pPIC9K-Chit was constructed successfully. The SDS-PAGE analysis showed that the Mr of Chitotriosidase was about 50 000. The concentration of the protein was 24 mg/L and the purity was 97%. Conclusion Chitotriosidase gene was expressed successfully in yeast Pichia pastoris. The obtaining of Chitotriosidase protein with natural structure will play an important role in the further study of Chitotriosidase.

关 键 词：毕赤酵母 壳三糖酶 真菌 几丁质 

分 类 号：Q786[生物学—分子生物学]