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Updated: Jan 17, 2026

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

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Published on: October 18, 2022

通过CRISPR/Cas9介导构建一个YPS基因缺陷的Komagataella phaffii菌株,以增强BIAP II的表达

Haichao Li¹, Ping Gui¹, Xiao Li¹

¹Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.

Yeast (Chichester, England)

|September 24, 2025

概括

科马格塔 (Komagataella phaffii) 会分泌牛肠性酸酶 (BIAP II),但蛋白质会降解它. 通过减少降解,禁用YPS1基因显著提高了BIAP II产量.

科学领域:

生物化学生物化学
分子生物学分子生物学
蛋白质表达方式

背景情况:

牛肠性酸酶II型 (BIAP II) 有较高的特定活性.
Komagataella phaffii用于重组BIAP II表达,但蛋白质分解降低了产量,并使净化复杂化.

研究的目的:

为了识别和减轻负责BIAP II降解的蛋白酶在komagataella phaffii.分泌期间分泌.
为了提高复合BIAP II的生产产量和纯度.

主要方法:

研究了分泌途径蛋白酶,特别是yapsin (Yps) 和Yps1在BIAP II降解中的作用.
产生了一种YPS1基因淘汰菌株,以评估其对BIAP II生产的影响.
利用CRISPR/Cas9技术创建了三重的雅普辛基因缺陷菌株.
开发了一种双色定量PCR (DC-qPCR) 方法,以有效地确定基因剂量.

主要成果:

BIAP II的蛋白质分解裂变主要由分泌蛋白酶介导,其中Yps1是关键酶.
破坏YPS1基因增加了BIAP II的产量2.5倍,降低了降解.
在BIAP II序列中确定了Lys137和Lys138之间的Yps1裂痕部位.
一种三重雅普辛基因缺陷菌株进一步降低了BIAP II蛋白解.
DC-qPCR方法提高了基因剂量的查效率和准确性.

结论:

针对像Yps1这样的特定蛋白酶是一种有效的策略,可以减少Komagataella phaffii的降解并增强异质蛋白质的产生.
通过CRISPR/Cas9介导的基因编辑提供了一个强大的工具,用于改造酵母菌株以改善蛋白质表达.
开发的DC-qPCR方法为重组蛋白质生产中的菌株查提供了强大的方法.

关键词:

在 BIAP II 中,在CRISPR的三重编辑中.这是一种DC-qPCR技术.科玛格塔贝拉 (Komagataella phaffii) 是一个有趣的植物.雅普辛蛋白质酶是什么意思

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