Construction of a novel synergistic system for production and recovery of secreted recombinant proteins by the cell surface engineering
Construction of a novel synergistic system for production and recovery of secreted recombinant proteins by the cell surface engineering
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DOI：
10.1007/s00253-007-0868-1
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发表时间：
2007-06-01
影响因子：
5
通讯作者：
Ueda, Mitsuyoshi
中科院分区：
文献类型：
--
作者：
Shibasaki, Seiji;Kawabata, Ai;Ueda, Mitsuyoshi
我们确定了共培养酵母细胞展示的ZZ-域和分泌的Fc融合蛋白是否可以是一种新的工具，用于回收分泌的重组蛋白。金黄色葡萄球菌蛋白A的ZZ结构域在GAL 1启动子的控制下展示在酿酒酵母的细胞表面上。菌株S.将在其表面上展示ZZ-结构域的酿酒酵母BY 4742细胞用于与产生融合到作为亲和标签的Fc片段的靶蛋白的细胞共培养。将增强型绿色荧光蛋白或根霉脂肪酶分别与人免疫球蛋白G的Fc片段的N端和C端基因融合。通过荧光激活细胞分选和酶法分析，证明这些融合蛋白在培养基中成功产生，并通过与展示ZZ结构域的细胞表面的亲和结合而回收。这些结果表明，ZZ结构域展示细胞和Fc融合蛋白分泌细胞可用于分泌重组蛋白的生产和回收的协同过程。
We determined whether the cocultivation of yeast cells displaying a ZZ-domain and secreting an Fc fusion protein can be a novel tool for the recovery of secreted recombinant proteins. The ZZ-domain from Staphylococcus aureus protein A was displayed on the cell surface of Saccharomyces cerevisiae under the control of the GAL1 promoter. Strain S. cerevisiae BY4742 cells displaying the ZZ-domain on their surface were used for cocultivation with cells that produce a target protein fused to the Fc fragment as an affinity tag. The enhanced green fluorescent protein or Rhizopus oryzae lipase was genetically fused to the N and C termini of the Fc fragment of human immunoglobulin G, respectively. Through analysis by fluorescence-activated cell sorting and enzymatic assay, it was demonstrated that these fusion proteins are successfully produced in the medium and recovered by affinity binding with the cell surface displaying the ZZ-domain. These results suggest that the ZZ-domain-displaying cell and Fc fusion protein-secreting cell can be applied to use in synergistic process of production and recovery of secreted recombinant proteins.