title: Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE.
abstract: Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.
authors.affiliation: Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Selangor, Malaysia.
authors.forename: Few Ne
authors.initials: FN
authors.lastname: Chew
authors.affiliation: Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor, Malaysia.|Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia.
authors.forename: Wen Siang
authors.initials: WS
authors.lastname: Tan
authors.affiliation: Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, Selangor, Malaysia.
authors.forename: Tau Chuan
authors.initials: TC
authors.lastname: Ling
authors.affiliation: Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Selangor, Malaysia.|Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia.
authors.forename: Beng Ti
authors.initials: BT
authors.lastname: Tey
mesh_terms: D018533:Biomass; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000302:isolation & purification*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins
references.citation: Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C., Science 1994, 263, 802-805.
references.citation: March, J. C., Rao, G., Bentley, W. E., Appl. Microbiol. Biot. 2003, 62, 303-315.
references.citation: Yakhnin, A. V., Vinokurov, L. M., Surin, A. K., Alakhov, Y. B., Protein Expres. Purif. 1998, 14, 382-386.
references.citation: Jain, S., Singh, R., Gupta, M. N., J. Chromatogr. A 2004, 1035, 83-86.
references.citation: Noubhani, A. M., Dieryck, W., Chevalier, S., Santarelli, X., J. Chromatogr. A 2002, 968, 113-120.
references.citation: Chiang, C. L., Chen, C. Y., Chang, L. W., J. Chromatogr. B 2008, 864, 116-122.
references.citation: Dalal, S., Raghava, S., Gupta, M. N., Biochem. Eng. J. 2008, 42, 301-307.
references.citation: Cabanne, C., Noubhani, A. M., Hocquellet, A., Dole, F., Dieryck, W., Santarelli, X. J., J. Chromatogr. B 2005, 818, 23-27.
references.citation: Zhuang, R., Zhang, Y., Zhang, R., Song, C., Yang, K., Yang, A., Jin, B., Protein Expres. Purif. 2008, 59, 138-143.
references.citation: McRae, S. R., Brown, C. L., Bushell, G. R., Protein Expres. Purif. 2005, 41, 121-127.
references.citation: Narahari, C. R., Eichhorn, L. R., Strong, J. C., Ramasubramanyan, N., Rao, G., Frey, D. D., Biotechnol. Progr. 2001, 17, 150-160.
references.citation: Deschamps, J. R., Miller, C. E., Ward, K. B., Protein Expres. Purif. 1995, 6, 555-558.
references.citation: Jain, S., Teotia, S., Gupta, M. N., Protein Expres. Purif. 2004, 36, 76-81.
references.citation: Harrison, S. T. L., Biotechnol. Adv. 1991, 9, 217-240.
references.citation: Ho, C. W., Tan, W. S., Yap, W. B., Ling, T. C., Tey, B. T., Biotechnol. Bioproc. Eng. 2008, 13, 577-583.
references.citation: Cho, T. H., Ahn, S. J., Lee, E. K., Bioseparation 2002, 10, 189-196.
references.citation: Lewis, U. J., Clark, M. O., Anal. Biochem. 1963, 6, 303-315.
references.citation: Stephens, R. E., Anal. Biochem. 1975, 65, 369-379.
references.citation: Saeed, S. A., Boyde, T. R. C., Prep. Biochem. 1980, 10, 445-462.
references.citation: Jansen, S., Kriegesmann, B., Jones, R., Brenig, B., Anal. Biochem. 1995, 228, 177-178.
references.citation: Galland-Irmouli, A. V., Pons, L., Lucon, M., Villaume, C., Mrabet, N. T., Gueant, J. L., Fleurence, J., J. Chromatogr. B 2000, 739, 117-123.
references.citation: Kastenholz, B., Anal. Lett. 2004, 37, 657-665.
references.citation: Lee, K. W., Tan, W. S., J. Virol. Methods 2008, 151, 172-180.
references.citation: Ornstein, L., Ann. N. Y. Acad. Sci. 1964, 121, 321-351.
references.citation: Davis, B. J., Ann. N. Y. Acad. Sci. 1964, 121, 404-427.
references.citation: Chew, F. N., Tan, W. S, Ling, T. C., Tan, C. S., Tey, B. T., Anal. Biochem. 2009, 384, 353-355.
references.citation: Bradford, M. M., Anal. Biochem. 1976, 72, 248-254.
references.citation: Laemmli, U. K., Nature 1970, 227, 680-685.
references.citation: Shimada, K., Shimahara, K., Agric. Biol. Chem. 1985, 49, 3605-3607.
references.citation: Tsong, T. Y., Biophys. J. 1991, 60, 297-306.
references.citation: Weaver, J. C., Chizmadzhev, Y. A., Bioelectrochem. Bioenerg. 1996, 41, 135-160.
references.citation: Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. H., EMBO J. 1982, 1, 841-845.
references.citation: Xie, T., Tsong, T. Y., Biophys. J. 1990, 58, 897-903.
references.citation: Orlowski, S., Mir, L. M., Biochim. Biophys. Acta 1993, 1154, 51-63.
references.citation: Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., Cormier, M. J., Gene 1992, 111, 229-233.
references.citation: Ishii, M., Kunimura, J. S., Penna, T. C. V., Cholewa, O., Int. J. Pharm. 2007, 337, 109-117.
references.citation: Retamal, C. A., Thiebaut, P., Alves, E. W., Anal. Biochem. 1999, 268, 15-20.
references.citation: Branco, A. T., Ferreira, B. S., Filho, G. A. S., Anal. Biochem. 2008, 381, 267-269.
references.citation: Kaplan, B., Pras, M., J. Chromatogr. B 2002, 769, 363-370.
references.citation: Wu, R. S., Stedman, J. D., West, M. H. P., Pantazis, P., Bonner, W. M., Anal. Biochem. 1982, 124, 264-271.