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首页> 外文期刊>Protein Expression and Purification >Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry
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Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry

机译：重组人GM2激活蛋白在昆虫细胞中的表达：质谱的纯化和鉴定

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The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme P-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZN5-His, which. was used for the constitutive expression in stably transformed Sf'21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by, sequential chromatography on Ni-NTA-agarose,and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1 L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the, structure (GlcNAc)(2)Fuc(Man)(3). (C) 2002 Elsevier Science (USA). All-rights reserved. [References: 21]
机译：GM2激活蛋白（GM2AP）是小的非酶辅助因子，可协助酶P-己糖胺酶A抑制神经节苷脂GM2的溶酶体降解。编码该糖蛋白的基因突变会导致致命的神经系统疾病，即GM2-神经节苷脂酶的AB变体。在本文中，我们描述了使用杆状病毒表达载体系统（BEVS）和基于非裂解质粒的昆虫细胞表达系统（InsectSelect）来在Sf21细胞中过量表达GM2AP。对于BEVS，将编码人GM2AP-前原蛋白的cDNA克隆到表达载体pAcMP3中。通过与线性化杆状病毒DNA共转染而产生的重组病毒被用于感染Sf21细胞。对于非裂解表达系统，将GM2AP的cDNA插入到载体pIZN5-His中。在稳定转化的Sf'21细胞中使用α-内含子进行组成型表达。如通过细胞培养上清液的免疫印迹分析所显示的，在两个表达系统中，GM2AP前体蛋白均有效地分泌到培养基中。在BEVS中表达后，通过在Ni-NTA-琼脂糖和Con A-Sepharose上进行顺序色谱法纯化GM2AP，从1 L细胞培养上清液中纯化蛋白的产量最高为9 mg。在稳定转化的昆虫细胞中表达后，分泌的蛋白质首先通过阳离子交换进行浓缩，并通过金属离子亲和色谱纯化，产量为0.1 mg / L细胞培养上清液。重组蛋白具有刺激己糖胺酶A催化的神经节苷脂GM2降解的能力，从而证明了其生物学活性，并通过ESI-TOF质谱法对其同质性和糖基化进行了评估。虽然BEVS中的蛋白质表达导致部分糖基化和部分非糖基化的蛋白质，但稳定转化的细胞仅产生糖基化的蛋白质。在两个表达系统中，发现糖基化是相同的并且对应于结构（GlcNAc）（2）Fuc（Man）（3）。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：21]

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