Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.
Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.
复制标题
眼促促细胞分裂原乳泌素编码序列中稀有密码子的保守区域 3 分组。
DOI：
10.1167/iovs.12-10740
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发表时间：
2013
影响因子：
4.4
通讯作者：
Laurie,GordonW
中科院分区：
文献类型：
--
作者：
McKown,RobertL;Raab,RonaldW;Kachelries,Patricia;Caldwell,Sara;Laurie,GordonW
目的：催泪素是泪液中的促分泌有丝分裂原，虽然是一种泪液蛋白，但它促进基底泪液和泪腺分泌。由于规模扩大与其在干眼治疗中的潜在用途有关，我们探索了各种诱变策略来改变新生乳蛋白的稳定性、溶解性和翻译效率，并发现了罕见人类密码子的3′聚类。定点突变的lacritin编码cDNA“pLAC”产生24个不同的非同义突变和13个同义突变。非同义突变改变了氨基酸的非极性，碱性或酸性侧链丝氨酸。同义突变逐步优化大肠杆菌中罕见或不常见的人类密码子，而不改变指定的氨基酸。这些变化通过测序和蛋白质生产进行验证，并通过“稀有密码子计算器”（RCC）进行分析。从Ensembl中提取非人灵长类和非灵长类催泪蛋白编码序列，并使用适合于每个物种的密码子使用通过RCC进行分析。上级的产量得到个别疏水残基或预测的盐桥的修改，这表明生产受到限制的催泪蛋白的稳定性。因此，消除稀有密码子增加产量的效果较差。重要的是，RCC分析人类、非人类灵长类动物（鼠狐猴）和非灵长类动物（猫、树）的泪蛋白编码序列显示罕见密码子的显著3′聚类，不像人类脂质运载蛋白-1和其他21个广泛表达的人类泪液基因。主要通过疏水或盐桥诱变来提高Lacritin蛋白质产量，通过消除稀有密码子来提高较少。稀有密码子的3′聚类在所有检查的lacritin直系同源物中是保守的。
Purpose.: Lacritin is a prosecretory mitogen in tears and, although a tear protein, it promotes basal tearing and lacrimal gland secretion. Since scale up is relevant to its potential use in the treatment of dry eye, we explored various mutagenic strategies to alter the stability, solubility, and translational efficiency of nascent lacritin, and discovered 3′ clustering of rare human codons.Methods.: Site-directed mutagenesis of lacritin coding cDNA “pLAC” generated 24 different nonsynonymous and 13 synonymous mutations. Nonsynonymous mutations altered amino acids with nonpolar, basic or acidic side chains to serine. Synonymous mutation progressively optimized human codons that are rare or uncommon in Escherichia coli without changing the amino acid specified. These changes were validated by sequencing and protein production, and analyzed via the “rare codon calculator”(RCC). Nonhuman primate and nonprimate lacritin coding sequences were extracted from Ensembl, and analyzed via RCC using codon usage appropriate for each species.Results.: Superior yields were obtained by modification of individual hydrophobic residues or a predicted salt bridge, suggesting that production was limited by lacritin stability. Accordingly, elimination of rare codons increased yields less effectively. Importantly, RCC analysis of human, nonhuman primate (mouse lemur) and nonprimate (cat, tree shrew) lacritin coding sequences revealed remarkable 3′ clustering of rare codons, unlike human lipocalin-1 and 21 other widely expressed human tear genes.Conclusions.: Lacritin protein yields were improved primarily by hydrophobic or salt bridge mutagenesis and less so by elimination of rare codons. The 3′ clustering of rare codons is conserved in all lacritin orthologs examined.