Zheng-Mei Li , Yan Lin , Cong-hui Luo , Qiu-Li Sun , Chun-Liu Mi , Xiao-yin Wang , Tian-Yun Wang
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引用次数: 0
摘要
在真核生物中，将小核糖体亚基定位到 mRNA 转录本上需要翻译起始位点上的 Kozak 元素。Kozak 元素的序列会影响蛋白质合成的翻译效率。然而，Kozak 序列的上游核苷酸是否会影响重组蛋白在中国仓鼠卵巢（CHO）细胞中的表达仍不清楚。为了找到提高重组蛋白在CHO细胞中表达的最佳序列，我们比较了100个CHO基因中ATG周围的-10至+4序列，并构建了具有不同翻译强度的扩展Kozak元件。以经典 Kozak 元件为对照，研究了优化的扩展 Kozak 元件对分泌型碱性磷酸酶（SEAP）和人血清白蛋白（HSA）基因的影响。结果表明，优化的扩展 Kozak 序列能提高重组蛋白在 CHO 细胞中的稳定表达水平。此外，研究还发现，重组蛋白表达水平的提高与转录水平的提高无关。综上所述，优化扩展 Kozak 元件可提高重组蛋白在 CHO 细胞中的表达，有助于构建 CHO 细胞的高效表达系统。
Optimization of extended Kozak elements enhances recombinant proteins expression in CHO cells
In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, −10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.
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