Bioprocessing Technology Institute, Agency for Science, Technology and Research-A*STAR, 20 Biopolis Way #06-01, Singapore 138668, Singapore.
J Biotechnol. 2013 Sep 10;167(3):326-33. doi: 10.1016/j.jbiotec.2013.07.011. Epub 2013 Jul 19.
The human interferon-gamma (IFN-γ) is a potential drug candidate for treating various diseases due to its immunomodulatory properties. The efficient production of this protein can be achieved through a popular industrial host, Chinese hamster ovary (CHO) cells. However, recombinant expression of foreign proteins is typically suboptimal possibly due to the usage of non-native codon patterns within the coding sequence. Therefore, we demonstrated the application of a recently developed codon optimization approach to design synthetic IFN-γ coding sequences for enhanced heterologous expression in CHO cells. For codon optimization, earlier studies suggested to establish the target usage distribution pattern in terms of selected design parameters such as individual codon usage (ICU) and codon context (CC), mainly based on the host's highly expressed genes. However, our RNA-Seq based transcriptome profiling indicated that the ICU and CC distribution patterns of different gene expression classes in CHO cell are relatively similar, unlike other microbial expression hosts, Escherichia coli and Saccharomyces cerevisiae. This finding was further corroborated through the in vivo expression of various ICU and CC optimized IFN-γ in CHO cells. Interestingly, the CC-optimized genes exhibited at least 13-fold increase in expression level compared to the wild-type IFN-γ while a maximum of 10-fold increase was observed for the ICU-optimized genes. Although design criteria based on individual codons, such as ICU, have been widely used for gene optimization, our experimental results suggested that codon context is relatively more effective parameter for improving recombinant IFN-γ expression in CHO cells.
人干扰素-γ（IFN-γ）由于其免疫调节特性，是一种有潜力的治疗各种疾病的候选药物。通过一种流行的工业宿主，中国仓鼠卵巢（CHO）细胞，可以有效地生产这种蛋白质。然而，由于在编码序列中使用了非天然的密码子模式，重组表达外源蛋白的效率通常不理想。因此，我们展示了一种新开发的密码子优化方法在 CHO 细胞中增强异源表达的应用，用于设计合成 IFN-γ编码序列。对于密码子优化，早期的研究建议根据选定的设计参数（如单个密码子使用（ICU）和密码子背景（CC））建立目标使用分布模式，主要基于宿主的高表达基因。然而，我们基于 RNA-Seq 的转录组分析表明，CHO 细胞中不同基因表达类别的 ICU 和 CC 分布模式相对相似，与其他微生物表达宿主大肠杆菌和酿酒酵母不同。这一发现通过在 CHO 细胞中体内表达各种 ICU 和 CC 优化的 IFN-γ得到了进一步证实。有趣的是，与野生型 IFN-γ相比，CC 优化的基因表达水平至少增加了 13 倍，而 ICU 优化的基因表达水平最大增加了 10 倍。虽然基于单个密码子的设计标准，如 ICU，已被广泛用于基因优化，但我们的实验结果表明，密码子背景是提高 CHO 细胞中重组 IFN-γ表达的相对更有效的参数。
