Continuous Monitoring of Enzymatic Reactions on Surfaces by Real-Time Flow Cytometry: Sortase A Catalyzed Protein Immobilization as a Case Study

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• 作者：Tobias Heck ; Phu-Huy Pham ; Frederik Hammes ; Linda Th枚ny-Meyer ; Michael Richter
• 刊名：Bioconjugate Chemistry
• 出版年：2014
• 出版时间：August 20, 2014
• 年：2014
• 卷：25
• 期：8
• 页码：1492-1500
• 全文大小：367K
• ISSN：1520-4812

文摘

Only a few techniques, such as quartz crystal microbalance and surface plasmon resonance spectroscopy, enable the analysis of dynamic processes on solid supports. Here we have developed a straightforward assay based on flow cytometry to continuously follow enzymatic reactions directly on microparticle surfaces. We applied this real-time flow cytometry (RT-FCM) approach to study the covalent immobilization of green-fluorescent protein (GFPuv) on triglycine-modified polystyrene microbeads by the transpeptidase sortase A (SrtA) from Staphylococcus aureus. Though commonly treated as functionally identical catalysts, the SrtA variants SrtA_螖59 and SrtA_螖25, in which the N-terminal amino acid residues 1鈥?9 and 1鈥?5 of the native enzyme are truncated, were shown to perform very differently with regard to this particular immobilization reaction. While SrtA_螖59 efficiently catalyzed the covalent attachment of GFPuv to the surface (as indicated by a linear increase of microbead fluorescence), SrtA_螖25 was essentially inactive. Besides the length of the N-terminal amino acid extension on the SrtA construct, the position of the hexahistidine tag at either the N- or C-terminus affected the efficiency of enzymatic protein immobilization. Apart from three enzyme variants containing the native core structure of SrtA, we also included three recently evolved mutants of SrtA in this comparative study. With these mutants we observed a rapid initial attachment of the GFPuv target protein to the microbeads. However, with proceeding reaction time, cleavage of the covalently immobilized target protein from the surface prevailed over the coupling reaction, consequently causing a decline of microbead fluorescence. In general, the RT-FCM approach used herein represents a powerful analytical tool for qualitative dynamic studies of many heterogeneous enzymatic reactions or other binding events that influence the fluorescence properties of microparticle surfaces.