Purification and characterization of human procolipase expressed in yeast cells
Purification and characterization of human procolipase expressed in yeast cells
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DOI：
10.1006/prep.1998.0873
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发表时间：
1998-06-01
影响因子：
1.6
通讯作者：
Lowe, ME
中科院分区：
文献类型：
--
作者：
Cordle, RA;Lowe, ME
我们报道了重组人原胶原酶在酵母中的成功、高效和大规模表达。利用人原胶原酶全长cDNA，分别用天然的人原胶原酶信号肽序列和酵母的信号肽序列进行构建。这些构建体用于转化酵母细胞，并进行表达。使用天然的人信号肽的原胶原酶只有极少的表达。当酵母信号肽与天然信号肽交换时，原胶酶的分泌旺盛。表达量大于30 mg/l。重组蛋白通过免疫亲和层析从培养基中纯化，高纯度的原胶原酶无蛋白水解降解，其活性和结合特性与组织纯化的人胰腺胶原酶没有区别。在酵母细胞中的表达为表达完整的、未经加工的重组野生型和突变的原胶原酶提供了一个有用的工具。(C) 1998学术出版社。
We report the successful, efficient, and large-scale expression of recombinant human procolipase in yeast. Using the full-length cDNA of human procolipase, constructs were made using either the native human procolipase signal peptide sequence or the signal peptide sequence of yeast. These constructs were used to transform yeast cells, and expression was followed. Only minimal expression was seen with the procolipase using the native human signal peptide. Robust secretion of the procolipase occurred when the yeast signal peptide was exchanged for the native signal peptide. Expression yielded more than 30 mg/liter. The recombinant protein was purified from the medium by immunoaffinity chromatography, The highly purified procolipase was free of proteolytic degradation and displayed activity and binding characteristics that were indistinguishable from those of tissue-purified human pancreatic colipase. Expression in yeast cells provides a useful tool for expressing intact, unprocessed recombinant wild-type and mutated procolipase. (C) 1998 Academic Press.