Kruse S, Kleineidam R G, Roggentin P, Schauer R
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
Protein Expr Purif. 1996 Jun;7(4):415-22. doi: 10.1006/prep.1996.0062.
A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.
编码产气荚膜梭菌A99“小”唾液酸酶同工酶的一个1.4kb基因，包括其自身的启动子，先前已克隆到大肠杆菌JM 101中并在其中表达。由于所有将该酶纯化至均一性的尝试均未成功，因此开发了一种新策略。通过PCR技术扩增结构基因，并将其插入质粒载体pQE-10中，从而在该蛋白质的N端转移了一个六组氨酸亲和标签（His6）。为了使唾液酸酶蛋白的蛋白水解降解降至最低，将该基因亚克隆到蛋白酶活性降低的大肠杆菌菌株BL21(DE3)pLys S中。与原始克隆相比，唾液酸酶的产量提高了约2.5倍。通过用溶菌酶处理细菌细胞释放出该酶，然后在Ni-次氮基三乙酸琼脂糖上进行金属螯合层析，使其在SDS-PAGE中达到明显的均一性。该42 kDa的蛋白质富集了62倍，产率为82%，比活性为280 U mg-1。从1升细菌培养物中获得了总量为1 mg的唾液酸酶。为了进行包括结晶实验在内的未来研究，用氨肽酶K从唾液酸酶上去除组氨酸亲和标签。然后通过离子交换层析将唾液酸酶与氨肽酶K分离，在标准条件下使用4-甲基伞形酮基-α-D-N-乙酰神经氨酸，总产率为83%，比活性为305 U mg-1。与野生型酶相比，两种形式（有或没有组氨酸标签）的唾液酸酶表现出相似的动力学性质。
