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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (1): 72-79    DOI: 10.13523/j.cb.2009043
综述     
酵母系统表达人表皮生长因子研究进展 *
石鹏程,纪晓俊()
南京工业大学生物与制药工程学院 南京 211806
Advances in Expression of Human Epidermal Growth Factor in Yeast
SHI Peng-cheng,JI Xiao-jun()
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
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摘要：

人源表皮生长因子是一种从人体内分离出的蛋白质,具有刺激细胞生长的作用,临床用途广泛。但由于天然来源有限,化学合成成本高,利用基因工程技术生产人表皮生长因子具有很好的前景。酵母系统作为一种典型的真核表达系统,在生产异源蛋白时能进行翻译后修饰,如糖基化、形成二硫键等,并能有效地将重组蛋白分泌到细胞外,有益于下游分离提取,在生产重组人表皮生长因子的研究中备受青睐。综述了利用酵母系统表达人表皮生长因子的研究进展,包括各种酵母表达系统在其中的应用,并分别比较其优势和劣势,在此基础上对未来的研究重点进行展望。
关键词： 人表皮生长因子;  酿酒酵母;  巴斯德毕赤酵母;  解脂耶氏酵母;  表达策略    
Abstract:

Human epidermal growth factor (hEGF) is a kind of protein isolated from the human body. It can stimulate the cell growth and thus has a wide range of clinical applications. The use of genetic engineering to produce hEGF has great economic prospects due to its limited natural source and high cost of chemical synthesis. Yeast expression system, as a typical eukaryotic expression system, can perform post-translational modifications, such as glycosylation, disulfide bond formation, etc., and can effectively secrete the proteins outside of the cell so that the recombinant protein can be separated from the extracellular medium easily. Therefore, yeast is especially favored in the research of producing recombinant hEGF. This article reviews the progress of producing hEGF in various yeast expression systems, including Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, etc., and on this base, their pros and cons were compared. The future prospects of using yeast expression to produce hEGF are discussed in light of the current progress, challenges, and trends in this field. Guidelines for future studies are finally proposed.
Key words: Human epidermal growth factor    Saccharomyces cerevisiae    Pichia pastoris    Yarrowia lipolytica    Expression strategy
收稿日期: 2020-09-27 出版日期: 2021-02-09
ZTFLH:  Q819  
基金资助: * 国家优秀青年科学基金(21922806);江苏省“六大人才高峰”计划(2018-SWYY-047)
通讯作者: 纪晓俊     E-mail: xiaojunji@njtech.edu.cn
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石鹏程, 纪晓俊. 酵母系统表达人表皮生长因子研究进展 *[J]. 中国生物工程杂志, 2021, 41(1): 72-79.

SHI Peng-cheng, JI Xiao-jun. Advances in Expression of Human Epidermal Growth Factor in Yeast. China Biotechnology, 2021, 41(1): 72-79.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2009043    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I1/72

图1  人表皮生长因子的一级结构
信号序列 hEGF产量(mg/L)
α-factor prepro 3.17
共有序列 0.59
共有序列(转化酶突变) 0.52
共有序列+6aa(KEX2蛋白酶切割位点) 0.57
转化酶突变+6aa(KEX2蛋白酶切割位点) 0.54
共有序列+19aa(无Asn连接糖基化位点) 3.39
共有序列+19aa(有Asn连接糖基化位点) 3.53
表1  合成信号序列的分泌效率[48]
宿主系统 方法 载体 效果 hEGF产量
(mg/mL)		 参考
Saccharomyces
cerevisiae		 构建了一个包含酵母GAPDH启动子、合成hEGF基因和酵母ADH-1终止子的质粒 pYEGF-2 在胞内表达出有活性的EGF 30.0 Urdea[44]
在hEGF基因的上游插入了酿酒酵母来源的α因子前序 pYaEGF-23 能将超过90%成熟的hEGF分泌到培养基中 4.0 Brake[45]
设计不同的前导序列 pSW6 提高了酿酒酵母中hEGF的分泌 3.2 Clements[48]
研究了hEGF的表达动力学 pYaEGF-25 筛选合适的培养基 13.0 Coppella[50]
对生理变量,培养基和生物反应器操作模式进行了综合研究 - 提高hEGF发酵过程的生产率 259.2 Valdes [51]
Pichia pastoris 人工合成表皮生长因子在甲基营养型酵母中的表达 Puc19-KαEGF51及 pAO815-EGF51 分泌完好生物活性和正确物理性质的hEGF 100 黄秉仁[52]
优化毕赤酵母表达hEGF时的培养条件 pPIC9K 分析培养基、甲醇浓度、pH和温度对生产的影响 2.27 Eissazadeh[57]
在毕赤酵母X33中表达hEGF并纯化 pPICZa A 成功诱导蛋白分泌并纯化 5.8 高云鹏[65]
Hansenula
polymorpha		 在多形汉逊酵母中表达hEGF基因 PMOX -EGF 敲除KEX1基因能减少hEGF的C端水解 2.5 Heo[59]
构建动力学模型,研究细胞生长和hEGF生产的pH依赖性 PMOX 在分批补料培养的诱导期中切换pH值可提高产量 80.0 Moon[60]
Yarrowia
lipolytica		 hEGF基因与碱性胞外蛋白酶的前导肽和proⅠ融合表达 pXEGF1 细胞分泌酸性蛋白酶使产量降低 0.1 Hamsa[63]
表2  人表皮生长因子在酵母系统中的表达
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