利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2  

Constitutive Expression of Human Goose-type Lysozyme 2 in Pichia pastoris Using the GAP Promoter

作　　者：黄鹏 阎丽萍 张宁 石金磊 HUANG Peng;YAN Li-ping;ZHANG Ning;SHI Jin-lei(College of Clinieal Medieine,Shanghai University of Medicine and Health Sciences,Shanghai 201318,China;Clinical Laboratory,Affiliated Central Hospital of Qingdao University,Qingdao 266042,China;College of Basie Medieine,Shanghai University of Medieine and Health Sciences,Shanghai 201318,China;Sehool of Life Science and Teehnology,ShanghaiTeeh University,Shanghai 201210,China)

机构地区：[1]上海健康医学院临床医学院,上海201318 [2]青岛大学附属中心医院,青岛266042 [3]上海健康医学院基础医学院,上海201318 [4]上海科技大学生命科学与技术学院,上海201210

出　　处：《中国生物工程杂志》2018年第10期55-63,共9页China Biotechnology

基　　金：上海市卫生计生委科研课题(201740161);上海市自然科学基金(15ZR1421800)资助项目

摘　　要：利用甘油醛三磷酸脱氢酶（glyceraldehydes-3-phosphatedehydrogenase,GAP）启动子在毕赤酵母中表达人鹅型溶菌酶2（human goose-type lysozyme 2,h LysG2）,并在小试规模建立一套有效的重组hLysG2（recombinant h LysG2,rh LysG2）生产工艺流程。根据毕赤酵母密码子偏爱性设计并人工合成hLysG2基因,将其连接至pGAPZαA质粒中,构建重组表达质粒pGAPZαA-h LysG2。将重组表达载体线性化后电转化毕赤酵母GS115感受态细胞,通过Zeocin抗性筛选获取高拷贝重组菌株,并在5L生物反应器中进行发酵培养。发酵60h后发酵液上清酶活性达到最高,发酵液上清经SDS-PAGE及Western blot检测证实rh LysG2得到表达。与诱导型表达相比,组成型表达发酵时间缩短了48h,上清中rhLysG2总活性提高了23.8%;使用甲壳素亲和层析和分子筛层析对rhLysG2进行纯化后,每升发酵液上清可纯化到187.4mg重组蛋白,纯化产物纯度达99.0%以上;浊度测定法分析显示,在p H 5.6、30℃和0.1mol/L Na＋的条件下,rhLysG2可达到最大酶活性13 500U/mg。利用GAP启动子在毕赤酵母中成功表达了高纯度和高活性的rh LysG2,避免了甲醇的使用,缩短了发酵时间,提高了蛋白产量,为将rhLysG2开发为新型抗耐药菌药物奠定了基础。This study aimed to achieve the constitutive expression of human goose-type lysozyme 2（ h LysG2） in Pichia pastoris using the glyceraldehyde-3-phosphate dehydrogenase（ GAP） promoter and to establish an efficient strategy for the production of recombinant h LysG2（ rhLysG2） on a bench scale. The hLysG2 gene was synthesized according to the codon usage preference of P. pastoris and cloned into pGAPZαA vector.The resulting pGAPZαA-hLysG2 plasmid was linearized and transformed into competent P. pastoris GS115,followed by Geneticin screening. The transformants with higher Geneticin resistance were selected to investigate the constitutive expression of rhLysG2 in P. pastoris. The lytic activity of rh LysG2 in the fermentation broth reached its peak after 60 h of cultivation. SDS-PAGE and Western blot analysis showed that rhLysG2 was successfully secreted into the fermentation broth. There were a 23. 8% increase in the total lytic activity and a48 h reduction in the cultivation time in comparison with those of the P. pastoris strain integrated with the pPIC9 K-hLysG2 plasmid. Using chitin affinity and size-exclusion chromatography,rhLysG2 was purified with a yield of 187. 4 mg/L of fermentation supernatant,above 99. 9% purity and a specific activity of 13 500 U/mg under the condition of p H 5. 6,0. 1 mol/L of Na＋,30℃. In conclusion,rh LysG2 was expressed at high level in P. pastoris by codon optimization and had in vitro bactericidal activity against some pathogenic bacteria,which has laid a solid foundation for its possible future pharmaceutical applications.

关 键 词：人鹅型溶菌酶2 毕赤酵母 GAP启动子 组成型表达 杀菌活性 

分 类 号：R392.33[医药卫生—免疫学] R392.11[医药卫生—基础医学]