Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

中文翻译：

---

密码子协调减少了细菌表达的恶性疟原虫蛋白质中的氨基酸错配并提高了它们的免疫原性。

密码子使用频率会影响蛋白质的结构和功能。密码子的使用频率可能会影响一级，二级和三级蛋白质的结构。密码子使用中特定物种的差异可能导致表达不佳，功能丧失，不溶性或截短。“密码子协调”使天然密码子使用频率与表达宿主的频率更紧密地对齐，特别是在推定的域间区段中，在该区段中较慢的翻译速率可能在蛋白质折叠中起作用。恶性疟原虫基因在大肠杆菌中的异源表达由于其富含AT的密码子偏倚和高度重复的DNA序列而成为一个挑战。在这里，密码子协调被应用于疟疾抗原CelTOS（Cel1-吨raversal蛋白质ö okinetes和小号porozoites）。CelTOS是一种高度保守的恶性疟原虫蛋白质，参与通过蚊子和脊椎动物宿主细胞的细胞穿越。热变性后可逆地重新折叠，使其成为理想的疟疾疫苗候选者。比较了恶性疟原虫CelTOS（CH- Pf CelTOS）密码子协调序列在大肠杆菌中表达的蛋白质与天然密码子序列（N- Pf（CelTOS）评估密码子使用对蛋白质表达水平，溶解度，产量，稳定性，结构完整性，CelTOS特异性mAb识别和小鼠免疫原性的影响。虽然预期翻译的蛋白质是相同的，但是由密码子协调的序列产生的翻译产物的螺旋含量不同，并且在质谱中显示较小的多肽分布，表明密码子协调的版本的异质性较低，氨基酸错配较少。疏水性至疏水性氨基酸的取代比其他任何一种都更常见。CH- Pf CelTOS诱导的抗体水平明显高于N- PfCelTOS；然而，在两种抗原之间未检测到IFN-γ或IL-4细胞应答的显着差异。