Article

Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing

Department of Biological Sciences, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore.
Bioorganic & Medicinal Chemistry Letters (Impact Factor: 2.42). 06/2005; 15(10):2447-51. DOI: 10.1016/j.bmcl.2005.03.079
Source: PubMed

ABSTRACT

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.

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    • "In general, the presence of a specific active group on the surface of a ligand protein is essential for its singlepointed attachment to the support. Many strategies have been explored to develop such specific coupling groups, such as (strept) avidin–biotin system [12], unnatural amino acids [13], and inteinmediated protein splicing [14]. However, sequence alterations are either needed or not reqiured, which depend on multistep operation and thus present an obstacle for their large-scale application. "
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    ABSTRACT: Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli. The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications.
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    • "It is interesting to note that the immobilized DsRed protein, which only has red fluorescence as a tetramer, retained its red fluorescence thereby indicating that its quaternary architecture was unaffected by the attachment to the PEGylated glass surface. Yao and co-workers have also used NCL and EPL, for the selective immobilization of N-terminally Cys-containing polypeptide (Lesaicherre et al. 2002b) and proteins (Girish et al. 2005) onto a-thioester coated glass slides. In this case, the polypeptide/proteins are site-specifically immobilized through their N-termini, which may be convenient in cases where the C-terminal immobilization, described earlier, affects the activity of the protein. "
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    ABSTRACT: Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.
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    • "Immobilization via expressed protein ligation has been successfully applied to a number of systems both directly [14], [15] and via the addition of affinity reagents biotin [16] or specific functional groups including azides [17], [18]. The use of intein-based systems provides a site-selective method but is often limited due to difficulties with expression of large target-intein fusion proteins and side reactions of the thiol based chemistry. "
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    ABSTRACT: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.
    Full-text · Article · Feb 2007 · PLoS ONE
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