Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.
Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.
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DOI：
10.1002/biot.201600696
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发表时间：
2017-05
影响因子：
4.7
通讯作者：
Cochran JR
中科院分区：
文献类型：
--
作者：
Lim S;Glasgow JE;Filsinger Interrante M;Storm EM;Cochran JR
酵母表面展示是一种成熟的蛋白质分析和工程化技术，涉及将感兴趣的蛋白质表达为与酵母Aga 2 p交配蛋白的N-或C-末端的遗传融合物。历史上，酵母展示的蛋白质变体的侧翼是肽表位标签，其使得能够使用荧光一抗或二抗对构建体表达进行流式细胞术测量。在这里，我们建立在这种技术的基础上，开发了一种新的酵母展示策略，该策略包括将两种不同的蛋白质融合到Aga 2 p，一种融合到N-末端，一种融合到C-末端。这种方法允许抗体片段、配体或受体直接偶联到荧光蛋白读数的表达，消除了对表位标签的抗体染色以定量酵母蛋白表达水平的需要。我们表明，该系统简化了酵母细胞表面上测量的蛋白质-蛋白质结合相互作用的定量。此外，我们表明，该系统有利于共同表达的生物缀合酶和其相应的肽底物上相同的Aga 2 p构建，使酶的表达和催化活性，以测量酵母的表面上。
Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.
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影响因子：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S
影响因子：
64.5
作者：
JULIUS, D;BRAKE, A;THORNER, J
通讯作者：
THORNER, J
DOI：
10.1073/pnas.1102561108
发表时间：
2011-08-09
影响因子：
11.1
作者：
Jones, Douglas S., II;Tsai, Ping-Chuan;Cochran, Jennifer R.
通讯作者：
Cochran, Jennifer R.
影响因子：
2.9
作者：
Boder, ET;Wittrup, KD
通讯作者：
Wittrup, KD
DOI：
10.1073/pnas.1101046108
发表时间：
2011-07-12
影响因子：
11.1
作者：
Chen, Irwin;Dorr, Brent M.;Liu, David R.
通讯作者：
Liu, David R.