YTHDF1蛋白对CHO-K1-GS-/-细胞系外源蛋白表达的增强作用

YTHDF1 enhances the expression of exogenous proteins in the CHO-K1-GS‒/‒ cell line

摘要:

YTH结构域家族蛋白1 (YTH domain family protein 1, YTHDF1)是一种RNA结合蛋白，属于N⁶-甲基腺嘌呤(N⁶-methyladenosine, m⁶A)修饰的阅读器，其通过特定的结构域来识别和结合RNA上的m⁶A修饰，从而执行相应的生物学功能。本研究旨在探讨YTHDF1蛋白对中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞系外源蛋白表达水平的影响，并评估其在CHO细胞表达系统优化中的应用潜力。首先采用CRISPR/Cas9技术敲除CHO-K1细胞的谷氨酰胺合成酶(glutamine synthetase, GS)编码基因从而得到CHO-K1-GS^?/?细胞系，再分析过表达YTHDF1蛋白对CHO-K1-GS^?/?细胞外源蛋白表达的增强作用，Western blotting、qPCR以及荧光显微镜观察发现过表达YTHDF1蛋白显著提高了细胞中人血清白蛋白、单链抗体和绿色荧光蛋白的表达水平，且未对细胞的长期增殖活力及存活率产生显著负面影响。此外，翻译抑制剂放线菌酮(cycloheximide, CHX)实验证实YTHDF1蛋白主要通过促进细胞中mRNA的翻译而增强蛋白表达。本研究证明YTHDF1蛋白能通过促进mRNA的翻译过程来增强细胞外源蛋白合成能力，为高效生物制药细胞株的构建提供了新的理论基础和技术方向。

Abstract:

YTH domain family protein 1 (YTHDF1) is an RNA-binding protein and belongs to the reader modified by N⁶-methyladenosine (m⁶A). It recognizes and binds to m⁶A modifications on RNA through specific domains, thereby performing corresponding biological functions. This study aims to explore the effects of YTHDF1 on the expression levels of exogenous proteins in the Chinese hamster ovary (CHO) cell line and evaluate its application potential in the optimization of the expression system of CHO cells. Firstly, the glutamine synthetase (GS) gene of CHO-K1 cells was knocked out by CRISPR/Cas9 to obtain the CHO-K1-GS^?/? cell line. The enhancement effects of YTHDF1 overexpression on the expression of exogenous proteins in CHO-K1-GS^?/? cells were further analyzed. Western blotting, qPCR, and fluorescence microscopy observations showed that the overexpression of YTHDF1 significantly up-regulated the expression levels of human serum albumin, single-chain antibody, and green fluorescent protein in cells. Moreover, this process did not have a significantly negative impact on the long-term proliferation and survival rate of the cells. In addition, the experiment with the translation inhibitor cycloheximide (CHX) confirmed that YTHDF1 mainly enhanced the protein expression by promoting translation in cells. This study demonstrates that YTHDF1 can enhance the cell’s ability to synthesize exogenous proteins by promoting the translation process of mRNA. This strategy provides a new theoretical basis and technical direction for constructing efficient cell lines for producing biopharmaceuticals.