人Hialuronidase-1(变体8)的亚克隆、表达和纯化

Adriana Dm Del Monaco, M. Hirata
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引用次数: 2

摘要

透明质酸是脊椎动物细胞外基质的主要成分。它是一种由透明质酸酶家族的酶水解的糖胺聚糖,参与调节重要的生物过程,如血管生成和血管通透性。为了开发这种酶的合成途径,我们的目标是获得含有基因变体8 Hyal-1编码序列的质粒。为了获得质粒插入,我们计划了两个亚克隆位点,分别针对Hyal-1密码子序列的5 'Bam H-1'和3 'Not-1。将插入物亚克隆到质粒pET28-a中,转染大肠杆菌Bl-21进行表达。IPTG诱导最佳表达时间为4小时,Western blotting证实蛋白表达。有一个45 kDa的蛋白,从而证实了Hyal-1的存在。在琼脂糖镍柱上进行纯化,得到的蛋白量较大,约为25μg/L。本研究建议的途径是高效获得Hyal-1重组蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Subcloning, expression and purification of Human Hialuronidase-1, variant 8.
Hyaluronic Acid, HA is a major component of the extracellular matrix of vertebrates. It is a glycosaminoglycan hydrolyzed by enzymes of the hyaluronidase family, involved in the regulation of important biological processes such as angiogenesis and vascular permeability. As interest in the development of a synthesis route for this enzyme, we aim to obtain a plasmid containing the coding sequence of gene variant 8 Hyal-1. To obtain the plasmid insert was planned and two restriction sites for sub-cloning site directed at the 5 'Bam H-1' and 3 'Not-1 in codon sequence of Hyal-1. The insert was sub-cloned into plasmid pET28-a, and transfected for expression in Escherichia coli Bl-21. The expression was induced by IPTG in best time of 4 hours and confirmation of protein expression was performed by Western blotting. There was a 45 kDa protein, thus confirming the presence of Hyal-1. Purification was performed on nickel agarose column to obtain a larger amount of the protein, approximately 25μg/L. The route suggested in this study was efficient attainment of Hyal-1 recombinant protein.
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