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


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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    • "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.
    International Journal of Peptide Research and Therapeutics 12/2008; 14(4):351-357. DOI:10.1007/s10989-008-9161-0 · 0.91 Impact Factor
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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.
    PLoS ONE 02/2007; 2(11):e1164. DOI:10.1371/journal.pone.0001164 · 3.23 Impact Factor
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    ABSTRACT: This chapter provides a description of the application of the finite element method to the simulation of the thermoforming process. The objective of thermoforming simulation is the provision of a rational means of mold design and to also allow for the design of “optimal” final parts using the minimum amount of material. This can be achieved by comparing the simulated behavior using various materials and mold configurations with varying process conditions. This eliminates the need to perform inefficient and expensive “trial-and-error” procedures. A “state-of-the-art” review of the finite element method in the simulation of thermoforming is given. The review covers the details of the membrane and thick sheet finite element formulations as well as non-linear elastic (Ogden, Mooney-Rivlin) and visco-elastic (K-BKZ) material constitutive relationships. Some examples, giving comparisons between simulation results and experimental values are also included. Good agreement was obtained between the predicted and measured thickness distributions. The results also indicate that the choice of mateiral model (non-linear elastic versus visco-elastic) must be done carefully if reliable predictions of the thickness distribution are to be achieved. For straight thermoforming into shallow molds a non-linear elastic model is suitable. However, when deep drawn forming, complex mold geometry or plugassistance is involved, a visco-elastic model is required to obtain accurate predictions.
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