有效表現天然蛋白質的改良式類泛素融合蛋白系統

對於在大腸桿菌中難以表現的重組蛋白，我們可以利用類泛素融合蛋白系統來增進其表現量。因為自然界中有小類泛素蛋白酶的存在，它會將小類泛素從融合蛋白上切除，如此一來更增進了此技術的效用。在打破大腸桿菌並取得上清液後，我們可以僅利用一根鎳離子層析管柱就完成融合蛋白的純化及類泛素切除的動作。經由以上的步驟，洗出液即是我們想要的天然蛋白質。如此單管柱方法適合發展成更高效率的蛋白純化平台。另外特別的一點是，這套新系統可以有效的表達，並迅速的純化一些難以表現蛋白質。我們便利用這套系統來純化大腸桿菌RecA的N端區域突變蛋白，並證實大腸桿菌RecA的N端區域在同源重組時有結合雙股核醣核酸的能力，並能促進下游三股交換反應的發生。

關鍵字

同源重組，三股交換，重組酵素，類泛素，融合蛋白

並列摘要

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni2+-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Especially, this new system was shown to be effective for expression, and rapid purification of several difficult-to-produce authentic proteins. Using this system, we purified the E. coli RecA N terminal mutant protein and reinvestigate the E. coli RecA N terminal domain (NTD), and suggest it is involved in dsDNA binding and promote the three-strand exchange reaction.

並列關鍵字

homologous recombination, three-strand exchange, RecA, SUMO, fusion protein

參考文獻

Butt, T.R., Edavettal, S.C., Hall, J.P., and Mattern, M.R. 2005. SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43: 1-9.

Catanzariti, A.M., Soboleva, T.A., Jans, D.A., Board, P.G., and Baker, R.T. 2004. An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci 13: 1331-1339.

Catic, A., Misaghi, S., Korbel, G.A., and Ploegh, H.L. 2007. ElaD, a Deubiquitinating protease expressed by E. coli. PLoS ONE 2: e381.

Chen, L.T., Ko, T.P., Chang, Y.C., Lin, K.A., Chang, C.S., Wang, A.H.J., and Wang, T.F. 2007. Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins. Nucleic Acids Res. 35: 1787-1801.

Chen, Y.K., Leng, C.H., Olivares, H., Lee, M.H., Chang, Y.C., Kung, W.M., Ti, S.C., Lo, Y.H., Wang, A.H., Chang, C.S., et al. 2004. Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation. Proc Natl Acad Sci U S A 101: 10572-10577.

國際替代計量

有效表現天然蛋白質的改良式類泛素融合蛋白系統

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