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首页> 外文期刊>Protein Expression and Purification >Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens
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Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens

机译：产气荚膜梭菌的重组α-N-乙酰半乳糖苷酶的纯化和鉴定

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摘要

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-D-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant NAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96 kDa and the native molecular weight of 72.42 kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C perfringens. With adequate expression in E coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells. (C) 2003 Elsevier Inc. All rights reserved. [References: 33]
机译：产气荚膜梭菌α-N-乙酰半乳糖苷酶（alphaNAG）水解产生血型A（2）抗原的H抗原，血型O的末端N-乙酰-α-D-半乳糖胺。血型O在ABO系统中普遍兼容。天然酶的纯化非常困难，产率很低。为了获得令人满意的产率的酶，将编码梭菌酶的基因克隆到大肠杆菌T7表达系统中。通过离子交换色谱法和高压液相色谱法从细胞裂解物中获得了高度纯化的重组NAG制剂。通过SDS-PAGE，最终制备物是均质的，分子量为71.96 kDa，天然分子量为72.42 kDa。该酶对末端N-乙酰半乳糖胺残基具有高度选择性。没有检测到其他显着的糖苷外切酶活性，特别是神经氨酸酶。酶的最适pH在6.5和7.0之间，并且活性不受离子强度的影响。 ELISA实验证明了针对血液A（2）型表位的活性。这些特征与产气荚膜梭菌的天然αNAG相似。在大肠杆菌中具有足够的表达，可以获得足够的重组alphaNAG酶质量，可用于将人类血液A（2）型红细胞酶促转化为普遍可输血的O型红细胞。 （C）2003 Elsevier Inc.保留所有权利。 [参考：33]

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