CLONING, EXPRESSION OF EcCSPA GENE AND PURIFICATION OF ITS PROTEIN
Main Article Content
Authors
A.S. Nizkorodova
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, The Science Committee, Ministry of Education and Science of the Republic of Kazakhstan, Almaty, Kazakstan
E.V. Polyanskaya
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, The Science Committee, Ministry of Education and Science of the Republic of Kazakhstan, Almaty, Kazakstan
A.M. Smagulov
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, The Science Committee, Ministry of Education and Science of the Republic of Kazakhstan, Almaty, Kazakstan
B.K. Iskakov
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, The Science Committee, Ministry of Education and Science of the Republic of Kazakhstan, Almaty, Kazakstan
Abstract
Cold-shock proteins (CSPs) are expressed in all living cells as the part of acclimation reaction to temperature decrease. These proteins are able to bind single stranded nucleic acids; it is also shown that some of cold-shock proteins are RNA chaperons. The mechanism of action during the cold-stress for these proteins is not completly understood yet. It’s thought that CSPs can inhibit expression of majority of cellular proteins via unspecific binding to their mRNAs during the cold-shock. On the other hand CSPs can act as RNA chaperons, which are thought to facilitate translation by destabilizing secondary structures occurring in mRNAs with decrease of temperature and interfering with their translation. To define CSP’s cell functions some in vitro experimental procedures are required, that presupposes existence of highly-purified and functionally active CSPs. Existing CSPs purification procedures are complicated, expensive and take long time. In this research we present one-stage CSP purification method, approved on EcCSPA. The procedure is based on affinity chromatography with usage of commercial nickel-nitrilotriacetate (Ni-NTA) agarose, which is able to specifically bind histidine sequences (His-tag). All protein purification procedure, including expression in bacterial cells, lasts one laboratory day. Applying this procedure for EcCSPA purification we have obtained native EcCSPA protein with purity 92,8% and concentration 2,9 g per liter of initial bacterial suspension.
Keywords
cold-shock proteins (CSP), EcCSPA, Ni-NTA agarose
Article Details
References
Phadtare S., Inouye M., Severinov K. The nucleic acid melting activity of Escherichia coli CspE is critical for transcription antitermination and cold acclimation of cells // The journal of biological chemistry. – 2002. – Vol. 277(9). – P. 7239-7245.
Jiang W., Hou Y., Inouye M. CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone // Journal of Biological Chemistry. – 1997. – Vol. 272(1). – P. 196-202.
Ermolenko D.N., Makhatadze G.I. Bacterial cold-shock proteins // CMLS. – 2002. – Vol. 59. – P. 1902-1913.
Hofweber R., Horn G., Langmann T., Balbach J., Kremer W., Schmitz G., Kalbitzer H.R. The influence of cold shock proteins on transcription and translation studied in cell-free model systems // FEBS Journal. – 2005. – Vol. 272. – P. 4691-4702.
Phadtare S., Severinov K. RNA remodeling and gene regulation by cold shock proteins // RNA Biology. – 2010. – Vol. 7(6). – P. 788-795.
Phadtare S., Severinov K. Comparative analysis of changes in gene expression due to RNA melting activities of translation initiation factor IF1 and a cold shock protein of the CspA family // Genes to Cells. – 2009. – Vol. 14. – P. 1227-1239.
Bae W., Xia B., Inouye M., Severinov K. Escherichia coli CspA-family RNA chaperones are transcription antiterminators // PNAS. – 2000. – Vol. 97(14). – P. 7784-7789.
Brandi A., Spurio R., Gualerzi1 C.O., Pon C.L. Massive presence of the Escherichia coli ‘major cold-shock protein’ CspA under non-stress conditions // The EMBO Journal. – 1999. – Vol. 18(6). – P. 1653-1659.
Schindelin H., Herrler M., Willimsky G., Marahiel M.A., Heinemann U. Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB // Proteins. – 1992. – Vol. 14. – P. 120-124.
Bae W., Phadtare S., Severinov K., Inouye M. Characterization of Escherichia coli cspE, whose product negatively regulates transcription of cspA, the gene for the major cold shock protein // Molecular Microbiology. – 1999. – Vol. 31. – P. 1429-1442.
Mayr B., Kaplan T., Lechner S., Scherer S. Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201 // Journal of Bacteriology. – 1996. – Vol. 178(10). – P. 2916-2925.
Vorackova I., Suchanova S., Ulbrich P., Diehl W.E., Ruml T. Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography // Protein Expression and Purification. – 2011. – Vol. 79. – P. 88-95.
Wingfield P.T. Preparation of soluble proteins from Escherichia coli // In: Current protocols in protein science. – 1995. – Vol. 1, New York: Wiley and Sons. – P. 6.2.1-6.2.15.
Gu J., Stephenson C.G., Iadarola M.J. Recombinant proteins attached to a Ni-NTA column: Use in affinity purification of antibodies // BioTechniques. – 1994. – Vol. 17. – P. 257-262.
Schägger H. Tricine-SDS-PAGE // National Protocols. – 2006. – Vol. 1. – P. 16-22.