Prokaryotic Expression and Purification of AtSPX1 Protein in Arabidopsis thaliana

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.013

Abstract

Abstract:

The proteins containing SPX domain exist widely in eukaryotes, and the functions of this kind of proteins are not clear yet, however some of them are involved in phosphorus signaling and some in iron signaling. SPX proteins in Arabidopsis thaliana can be divided into 4 families. AtSPX1 used in this paper belongs to the family containing only SPX domain, while other 3 families containing extra gene sequences. Phylogenetic analysis showed that amino acid encoded by AtSPX1 was close with dicots in sequence, but distant from monocots. In order to reveal the relationship between structural property and biological function, we carried out solubility expression experiments of the proteins in vitro, constructed prokaryotic expression vector in vitro, and had the high soluble expression of the protein in Escherichia coli’s cells. The expressed His-tag proteins allowed the purification more convenient, i.e, the inserted SUMO fusion protein tag could be digested by protease, and the target protein could be purified by ammonium sulfate precipitation. The further purification was completed using size exclusion chromatographic column, which yielded a profile corresponding to a monomeric AtSPX1 in solution. This work provided a strategy for the purification of AtSPX1 protein.

Key words: SPX domain, prokaryotic expression, protein purification, Arabidopsis thaliana

Hu Tao, An Yan, Lv Qundan, Xu Yingwu. Prokaryotic Expression and Purification of AtSPX1 Protein in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2015, 31(8): 88-93.

References

[1] Spain BH, Koo D, Ramakrishnan M, et al. Truncated forms of a novel yeast protein suppress the lethality of a G protein alpha subunit deficiency by interacting with the beta subunit[J]. J Biol Chem, 1995, 270（43）：25435-25444.
[2] Creasy CL, Madden SL, Bergman LW. Molecular analysis of the PHO81 gene of Saccharomyces cerevisiae[J]. Nucleic Acids Res, 1993, 21（8）：1975-1982.
[3] Battini JL, Rasko JE, J Miller AD. A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses：possible role in G protein-coupled signal transduction[J]. Proc Natl Acad Sci USA, 1999, 96（4）：1385-1390.
[4] Duan K, Yi K, Dang L, et al. Characterization of a sub-family of Arabidopsis genes with the SPX domain reveals their diverse functions in plant tolerance to phosphorus starvation[J]. Plant J, 2008, 54（6）：965-975.
[5] Wang C, Huang W, Ying Y, et al. Functional characterization of the rice SPX-MFS family reveals a key role of OsSPX-MFS1 in controlling phosphate homeostasis in leaves[J]. New Phytologist, 2012, 196（1）：139-148.
[6] Bun-Ya M, Harashima S, Oshima Y. Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae[J]. Mol Cell Biol, 1992, 12（7）：2958-2966.
[7] Ogawa N, DeRisi J, Brown PO. New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis[J]. Mol Biol Cell, 2000, 11（12）：4309-4321.
[8] Ghillebert R, Swinnen E, De Snijder P, et al. Differential roles for the low-affinity phosphate transporters Pho87 and Pho90 in Saccharomyces cerevisiae[J]. Biochem J, 2011, 434：243-251.
[9] Wang Y, Ribot C, Rezzonico E, et al. Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis[J]. Plant Physiol, 2004, 135（1）：400-411.
[10] Wang C, Ying S, Huang H, et al. Involvement of OsSPX1 in phosphate homeostasis in rice[J]. Plant J, 2009, 57（5）：895-904.
[11] Zhao L, Liu F, Xu W, et al. Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana[J]. Plant Biotechnol J, 2009, 7（6）：550-561.
[12] Wang C, Wei Q, Zhang K, et al. Down-Regulation of OsSPX1 causes high sensitivity to cold and oxidative stresses in rice seedlings[J]. PloS One, 2013, 8（12）：e81849.
[13] Liu F, Wang Z, Ren H, et al. OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice[J]. Plant J, 2010, 62（3）：508-517.
[14] Yao ZF, Liang CY, Zhang Q, et al. SPX1 is an important component in the phosphorus signalling network of common bean regulating root growth and phosphorus homeostasis[J]. Journal of Experimental Botany, 2014, 65：3299-3310.
[15] Shi J, Hu H, Zhang K, et al. The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice[J]. Journal of Experimental Botany, 2014, 65（3）：859-870.
[16] Puga MI, Mateos I, Charukesi R, et al. SPX1 is a phosphate-dependent inhibitor of PHOSPHATE STARVATION RESPONSE 1 in Arabidopsis[J]. Proc Nat Acad Sci USA, 2014, 111（41）：14947-14952.
[17] Lv Q, Zhong Y, Wang Y, et al. SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice[J]. Plant Cell, 2014, 26（4）：1586-1597.
[18] Wang Z, Ruan W, Shi J, et al. Rice SPX1 and SPX2 inhibit phosphate starvation responses through interacting with PHR2 in a phosphate-dependent manner[J]. Proc Natl Acad Sci USA, 2014, 111（41）：14953-14958.
[19] Zhang F, Xu Y, Zhang Z. Heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of KDO8P synthase from Arabidopsis thaliana[J]. Protein Expression and Purification, 2014, 101：133-137.