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Site-specific protein and peptide immobilization on a biosensor surface by pulsed native chemical ligation.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Helms, Brett A. Baal, Ingrid Van Merkx, Maarten Meijer, E. W. |
| Copyright Year | 2007 |
| Abstract | The site-specific immobilization of proteins and peptides on a surface represents a significant challenge because many proteomics applications require strategies that preserve the innate functionality of these biomolecules at interfaces. Unfortunately, routine protocols for protein immobilization usually result in the heterogeneous presentation of the ligand at the solid–liquid interface. As a result, the data that are obtained from analyte-binding experiments sometimes yield results that conflict with the analogous data that were obtained in solution. In recent years, this deficiency has been addressed by using chemoselective strategies that are based on known bioorthogonal reactions such as the native chemical ligation, the Staudinger ligation or the Huisgen azide– alkyne cycloaddition. While these reactions have been used extensively for the construction of protein microarrays, the modified substrates have not been amenable to any real-time kinetic analysis of protein function. Because many protein interactions in vivo are under kinetic control, new metholodologies to obtain this kind of information are highly desirable. Advanced techniques like surface plasmon resonance (SPR) routinely provide kinetic as well as thermodynamic data for protein interactions at surfaces; this is in large part due to instrumentation like the Biacore platform, which uses a microfluidic set-up to monitor biomolecular interactions. To the best of our knowledge, the aforementioned chemoselective ligation methods have not been applied to the modification of biosensor surfaces for SPR. In this communication, we describe the facile and direct site-specific immobilization of proteins and peptides on a biosensor surface by using the native chemical ligation (NCL) reaction. This protocol allows control of both the orientation of the ligand presented at the surface, as well as its density in a highly programmable manner. Our approach, while general, is designed for use in a Biacore instrument, which facilitates the evaluation of the methodology through SPR. Our experimental design required a biosensor surface with N-terminal cysteines for direct conjugation to proteins and peptides that bear C-terminal thioesters by NCL (Figure 1). The biosensor surfaces that are typically used in the Biacore apparatus are constructed on a microfluidic chip, and consist of a |
| File Format | PDF HTM / HTML |
| DOI | 10.1002/cbic.200700355 |
| PubMed reference number | 17763488 |
| Journal | Medline |
| Volume Number | 8 |
| Issue Number | 15 |
| Alternate Webpage(s) | http://www.mate.tue.nl/mate/pdfs/8537.pdf |
| Alternate Webpage(s) | https://doi.org/10.1002/cbic.200700355 |
| Journal | Chembiochem : a European journal of chemical biology |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
