在真核生物中,小核糖体亚基在mRNA转录物中的定位需要在起始位点翻译Kozak元件。Kozak元件的序列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影响中国仓鼠卵巢(CHO)细胞中重组蛋白的表达尚不清楚。为了寻找增强重组蛋白在CHO细胞中表达的最佳序列,比较了100个CHO基因中ATG周围的-10到+4个序列,构造了具有不同平移强度的扩展Kozak元件。使用经典的Kozak元素作为控件,研究了优化的延伸Kozak元件对分泌的碱性磷酸酶(SEAP)和人血清白蛋白(HSA)基因的影响。结果表明,优化后的延伸Kozak序列可以增强重组蛋白在CHO细胞中的稳定表达水平。此外,发现重组蛋白表达水平的增加与较高的转录水平无关。总之,优化延伸Kozak元件可以增强重组蛋白在CHO细胞中的表达,有助于CHO细胞高效表达系统的构建。
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.