Abstract The gene encoding the amylolytic enzyme Amo45, originating from a metagenomic project, was retrieved by a consensus primer-based approach for glycoside hydrolase (GH) family 57 enzymes. Family 57 contains mainly uncharacterized proteins similar to archaeal thermoactive amylopullulanases. For characterization of these family members soluble, active enzymes have to be produced in sufficient amounts. Heterologous expression of amo45 in E.coli resulted in low yields of protein, most of which was found in inclusion bodies. To improve protein production and to increase the amount of soluble protein, two different modifications of the gene were applied. The first was fusion to an N-terminal His-tag sequence which increased the yield of protein, but still resulted in high amounts of inclusion bodies. Co-expression with chaperones enhanced the amount of soluble protein 4-fold. An alternative modification was the attachment of a peptide consisting of the amino acid sequence of the mobile-loop of the co-chaperonin GroES of E.coli. This sequence improved the soluble protein production 5-fold compared to His6-Amo45 and additional expression of chaperones was unnecessary.

中文翻译：

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通过与辅助伴侣蛋白 GroES 的移动环区融合在大肠杆菌中改善编码淀粉分解酶 Amo45 的基因的可溶性表达

摘要 编码淀粉分解酶 Amo45 的基因源自宏基因组计划，通过基于共有引物的方法检索糖苷水解酶 (GH) 家族 57 酶。家族 57 主要包含与古细菌热活性淀粉支链淀粉酶相似的未表征蛋白质。为了表征这些家族成员的可溶性活性酶，必须以足够的量生产。amo45 在大肠杆菌中的异源表达导致蛋白质产量低，其中大部分在包涵体中发现。为了提高蛋白质产量和增加可溶性蛋白质的量，应用了两种不同的基因修饰。第一个是与 N 端 His 标签序列融合，这增加了蛋白质的产量，但仍然导致大量的包涵体。与分子伴侣的共表达使可溶性蛋白质的量增加了 4 倍。另一种修饰是连接由大肠杆菌共伴侣蛋白 GroES 的移动环的氨基酸序列组成的肽。与 His6-Amo45 相比，该序列将可溶性蛋白质的产量提高了 5 倍，并且不需要额外的分子伴侣表达。