Receptor-mediated endocytosis as a selection force to enrich bacteria expressing rhodostomin on their surface
Receptor-mediated endocytosis as a selection force to enrich bacteria expressing rhodostomin on their surface
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DOI：
10.1007/bf02255917
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发表时间：
2000-01-01
影响因子：
11
通讯作者：
Lo, SCJ
中科院分区：
文献类型：
--
作者：
Chang, HH;Shih, KN;Lo, SCJ
以前，我们开发了一种在大肠杆菌外表面表达去整合素-蛇毒罗多斯明(Rho)的Trat展示系统[J Bied Sci 6：-70；1999]。为了展示Trat展示系统的新的潜在用途，我们采用生物素标记技术，结合十二烷基硫酸钠-PAGE和流式细胞仪分析，进一步证明和确认了Trat-Rho在大肠杆菌表面的表达。我们还发现，细胞表面Trat-Rho的表达不仅促进了细菌与BHK-21细胞的黏附，而且诱导了细菌内化到BHK-21细胞中。这一特性使我们能够从Trat-Rho细菌和β-半乳糖苷酶阳性的Babe的混合物开始，通过四次BHK-21细胞吞噬和琼脂平板上吞噬细菌的复制，以10(2)：10(7)的比例重组RIA，使Trat-Rho表达细菌丰富约10,000倍，因此，我们建议可以应用Trat展示系统，通过受体介导的吞噬和再扩增循环，从大量展示文库中筛选出表达特定肽序列的细菌。版权所有(C)2000国家科学委员会，中华民国和S.Karger AG，巴塞尔。
Previously, we developed a TraT display system to express snake venom rhodostomin (RHO), a disintegrin, on the external surface of Escherichia coli [J Biomed Sci 6:64-70;1999], To show a new potential use of the TraT display system, we employed a biotin labeling technique coupled with SDS-PAGE and flow cytometry analyses to further demonstrate and confirm the expression of TraT-RHO on the E,coli surface. We also showed that the expression of TraT-RHO on the cell surface not only facilitated the bacteria adhesion to BHK-21 cells but also induced bacterial internalization into BHK-21 cells. This feature allowed us to enrich the TraT-RHO expression bacteria about 10,000-fold starting with a mixture of TraT-RHO bacteria with beta-galactosidase-positive babe; ria in a ratio of 10(2):10(7) through four cycles of BHK-21 cell endocytosis and replating of engulfed bacteria on agar plates, We therefore suggest that the TraT display system can be applied to select out bacteria expressing a specific peptide sequence from a large population of display library through the process of receptor-mediated endocytosis and reamplification cycles. Copyright (C) 2000 National Science Council, ROC and S. Karger AG, Basel.