Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display
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- 作者：Carrie J. Marshall ; Nitin Agarwal ; Jeet Kalia ; Vanessa A. Grosskopf ; Nicholas A. McGrath ; Nicholas L. Abbott ; Ronald T. Raines ; Eric V. Shusta
- 刊名：Bioconjugate Chemistry
- 出版年：2013
- 出版时间：September 18, 2013
- 年：2013
- 卷：24
- 期：9
- 页码：1634-1644
- 全文大小：466K
- 年卷期：v.24,no.9(September 18, 2013)
- ISSN：1520-4812
文摘
Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide鈥?/i>alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.