[1]张 念,林颖颖,祁可丹,等.草酸青霉16 β-葡萄糖苷酶的异源表达[J].江西师范大学学报(自然科学版),2023,(03):237-241.[doi:10.16357/j.cnki.issn1000-5862.2023.03.03]
 ZHANG Nian,LIN Yingying,QI Kedan,et al.The Heterologous Expression of Penicillium oxalicum 16 β-Glucosidase[J].Journal of Jiangxi Normal University:Natural Science Edition,2023,(03):237-241.[doi:10.16357/j.cnki.issn1000-5862.2023.03.03]
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草酸青霉16 β-葡萄糖苷酶的异源表达()
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《江西师范大学学报》(自然科学版)[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2023年03期
页码:
237-241
栏目:
出版日期:
2023-05-25

文章信息/Info

Title:
The Heterologous Expression of Penicillium oxalicum 16 β-Glucosidase
文章编号:
1000-5862(2023)03-0237-05
作者:
张 念林颖颖祁可丹王国平欧阳蓓赵喜华*
(江西师范大学生命科学学院,江西 南昌 330022)
Author(s):
ZHANG NianLIN YingyingQI KedanWANG GuopingOUYANG BeiZHAO Xihua*
(College of Life Science, Jiangxi Normal University,Nanchang Jiangxi 330022,China)
关键词:
β-葡萄糖苷酶 异源表达 酶活
Keywords:
β-glucosidase heterologous expression enzymatic activity
分类号:
Q 812
DOI:
10.16357/j.cnki.issn1000-5862.2023.03.03
文献标志码:
A
摘要:
为探究真核菌株草酸青霉16的β-葡萄糖苷酶(16BGL)在不同宿主中的异源表达情况,该文通过基因克隆技术,将16BGL基因分别与pPIC9K、pET-28a、pGAPZαA载体进行连接,转化到毕氏酵母GS115或大肠杆菌Rosetta(DE3)上,筛选阳性克隆子,比较并分析其表达情况.结果表明:16BGL-9K未能成功电转到毕氏酵母GS115上; 将16BGL-28a转化到大肠杆菌Rosetta上,使用最终物质的量浓度为50 μmol·L-1的IPTG和质量分数为1.7%的乳糖在22 ℃下诱导可实现高水平表达,但极易形成包涵体且无活性.为了消除包涵体的影响,在相同条件下将16BGL的N端加入DsbA分泌信号肽,经SDS-PAGE检验,16BGL有高水平可溶性表达,但无活性; 将16BGL-ZαA电转到GS115上,筛选阳性克隆子并接种到含YPG培养基的摇瓶中,结果表明:上清液蛋白质量浓度达到23.723 mg·mL-1,且有活性.真核来源的16BGL基因适合于真核宿主表达.
Abstract:
The heterologous expression of β-glucosidase(16BGL)of a eukaryotic strain Penicillium oxalicum 16 in different hosts is explored.The 16BGL gene is ligated with pPIC9K, pET-28a,and pGAPZαA respectively, and transformed into Pichia pastoris GS115 or Escherichia coli Rosetta(DE3), the positive clones are screened, and their expression is compared and analyzed.16BGL-9K can not be successfully transferred into GS115 by electroporation.16BGL-28a in Rosetta achieves a high level of expression after induction at 22 ℃ with IPTG at a final concentration of 50 μmol·L-1 and 1.7% lactose, but it is very easy to form inclusion bodies without activity.In order to eliminate the influence of inclusion bodies, a secretion signal peptide DsbA is added in the N-terminal of 16BGL, 16BGL achieves a high level of soluble expression under the same conditions, but no activity is detected by SDS-PAGE.16BGL-ZαA is transferred to GS115, the positive clones are screened and inoculated into YPG, and the supernatant possesses a protein concentration of 23.723 mg·mL-1 and shows an enzymatic activity.The eukaryotic gene 16BGL is suitable for expression in eukaryotic hosts.

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备注/Memo

备注/Memo:
收稿日期:2022-11-16
基金项目:国家自然科学基金(22068014,21666010),江西省自然科学基金(GJJ11071)和江西师范大学博士科研启动基金(5451)资助项目.
通信作者:赵喜华(1977—),男,江西玉山人,教授,博士,主要从事酶工程和合成生物学研究.E-mail:xhzhao@jxnu.edu.cn
更新日期/Last Update: 2023-05-25