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通过Cre/loxP系统回收可选标记物,用于构建共同表达多种蛋白质的Komagataella phaffii菌株

Weixian Wang¹, Minghai Han², Guofei Zhu³

¹Analytical and Testing Center, Guizhou Institute of Technology, Guiyang, 550000, China.

Biotechnology letters

|February 28, 2024

概括

本研究提出了一个简单的Cre/loxP系统,用于回收Komagataella phaffii酵母中的可选择标记物. 这种方法通过顺序基因整合,使多种蛋白质能够有效地共同表达.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
酵母遗传学酵母遗传学

背景情况:

在Komagataella phaffii中共同表达多种蛋白质对于各种生物技术应用至关重要.
需要有效的顺序基因整合和可选择标记物循环利用方法来简化菌株构造.

研究的目的:

使用Cre/loxP系统开发一个方便的回收可选标记物的策略.
构建能够共同表达多种蛋白质的 Komagataella phaffii 菌株.

主要方法:

用lox71-Sh ble-lox66和一个向蛋白质基因构建了等离子体,转化为K. phaffii KM71.1.
整合了一种表达CRE复合酶的辅助等离子体,以促进Cre/loxP介导的重组.
甲醇诱导触发了CRE的产生,导致标记物回收和随后的序列基因插入.

主要成果:

该Cre/loxP系统成功回收了ZeoR及其标记物.
两个蛋白质基因被顺序集成到K. phaffii染色体中.
工程酵母菌株证明了多种蛋白质的成功联合表达.

结论:

开发的方法为可选择标记物回收提供了一种有效且易于操纵的策略.
这种方法促进了Komagataella phaffii中的多种蛋白质的染色体共同表达.
该策略简化了用于复杂蛋白质生产的酵母菌株的构建.

关键词:

科玛格塔贝拉 (Komagataella phaffii) 是一个有趣的植物.这就是Pichia pastoris.同表达多种蛋白质.克雷洛克斯 (Cre/loxPP) 是一个植物.可以选择的标记.

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