Abstract
The postgenome era has led to a new frontier of proteomics that requires the development of protein microarray, which enables us to unravel the biological function of proteins in a massively parallel fashion. Several ways of immobilizing proteins onto surfaces have been reported, but many of these attachments are unspecific, resulting in the unfavorable orientation of the immobilized proteins. His6 tag has been used to site-specifically immobilize proteins onto nickel-coated slides, which presumably oriented proteins uniformly on the surface of the slide. However, the binding between Ni-NTA and His tag proteins is not strong, causing the immobilized proteins to dissociate from the slide even under simple wash conditions. The authors have developed a novel strategy of using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. Array-scan results not only show successful immobilization of proteins onto slides by antibody detection method but also full retention of biological activities of the immobilized proteins. The strong and specific interaction between biotin and avidin also permits the use of stringent incubation and washing conditions on the protein microchip, thus making it a highly robust method for array studies.
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References
Chen, G. Y. J., Uttamchandani, M., Lue, Y. P. R., et al. (2003) Array-based technologies and their applications in proteomics. Curr. Top. Med. Chem. 3, 705–724.
Haab, B. B., Dunham, M. J., and Brown, P. O. (2001) Protein microarray for highly parallel detection and quantitation of specific proteins and antibodies in complex solution. Genome Biol. 2, 1–13.
Schweitzer, B. and Kingsmore, S. (2002) Measuring proteins on microarray. Curr. Opin. Biotechnol. 13, 14–19.
MacBeath, G. and Schreiber, S. L. (2000) Printing proteins as microarrays for high-throughput function determination. Science 289, 1760–1763.
Zhu, H., Klemic, J. F., Chang, S., et al. (2000) Analysis of yeast protein kinases using protein chips. Nat. Genet. 26, 283–289.
Wang, D., Liu, S., Trummer, B. J., et al. (2002) Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host. Nat. Biotechnol. 20, 275–280.
Zhu, H., Bilgin, M., Bangham, R., et al. (2001) Global analysis of protein activities using proteome chips. Science 293, 2101–2105.
Lesaicherre, M. L., Uttamchandani, M., Chen, G. Y. J., et al. (2002) Developing sitespecific immobilization strategies of peptides in a microarray. Bioorg. Med. Chem. Lett. 12, 2079–2083.
Lesaicherre, M. L., Uttamchandani, M., Chen, G. Y. J., et al. (2002) Antibody-based fluorescence detection of kinase activity on a peptide array. Bioorg. Med. Chem. Lett. 12, 2085–2088.
Lesaicherre, M. L., Lue, Y. P. R., Chen, G. Y. J., et al. (2002) Intein-mediated biotinylation of proteins and its application in a protein microarray. J. Am. Chem. Soc. 124, 8768–8769.
Paborsky, L. R., Dunn, K. E., Gibbs, C. S., et al. (1996) A nickel chelate microtiter plate assay for six histidine-containing proteins. Anal. Biochem. 234, 60–65.
Cronan, J. E. (1990) Biotinylation of proteins in vivo. A posttranslational modification to label, purify, and study proteins. J. Biol. Chem. 265, 10,327–10,333.
Samols, D., Thornton, C. G., Murtif, V. L., et al. (1988) Evolutionary conservation among biotin enzymes. J. Biol. Chem. 263, 6461–6464.
Stolz, J., Ludwig, A., and Sauer, N. (1998) Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation. FEBS Lett. 440, 213–217.
Beckett, D., Kovaleva, E., and Schatz, P. J. (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929.
Cronan, J. E. and Reed, K. E. (2000) Biotinylation of proteins in vivo: a useful posttranslational modification for protein analysis. Methods Enzymol. 326, 440–458.
Cull, M. G. and Schatz, P. J. (2000) Biotinylation of proteins in vitro and in vivo using small peptide tags. Methods Enzymol. 326, 430–440.
Xu, M. Q. and Evan, T. C. (2001) Intein-mediated ligation and cyclization of expressed proteins. Methods 24, 257–277.
Evan, T. C. and Xu, M. Q. (1999) Intein-mediated protein ligation: harnessing nature’s escape artists. Biopolymers 51, 333–342.
Muir, T. W., Sondhi, D., and Cole, P. A. (1998) Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. USA 98, 6705–6710.
Muir, T. W. (2001) Development and application of expressed protein ligation. Synlett 6, 733–740.
Tolbert, T. and Wong, C.-H. J. (2000) Intein-mediated synthesis of proteins containing carbohydrates and other molecular probes. J. Am. Chem. Soc. 122, 5421–5428.
Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.
Sam brook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Lue, R.Y.P., Chen, G.Y.J., Zhu, Q., Lesaicherre, ML., Yao, S.Q. (2004). Site-Specific Immobilization of Biotinylated Proteins for Protein Microarray Analysis. In: Fung, E.T. (eds) Protein Arrays. Methods in Molecular Biology, vol 264. Humana Press. https://doi.org/10.1385/1-59259-759-9:085
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DOI: https://doi.org/10.1385/1-59259-759-9:085
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