The development of effective overproduction and purification approaches for ExuR transcription factor of Escherichia coli using affine chromatography
Keywords:
affine chromatography, cloning, transcription factors, ExuR, E. coli.
Abstract
A method for isolation and purification of the recombinant ExuR protein from Escherichia coli was
developed. Process includes cloning of the exuR gene into pGEMEX1 vector, followed by overproduction of
soluble protein in BL21 *(DE3) E. coli cells and a series of affine chromatography cycles on a column with
Ni-NTA agarose charged with nickel sulfate. This approach allows obtaining of electrophoretically
homogenious and functionally active ExuR protein for further investigation.
Downloads
Download data is not yet available.
References
1.Salgado H., Peralta-Gil M., Gama-Castro S., Santos-Zavaleta A., Muñiz-Rascado L.,
García-Sotelo J.S. et al RegulonDB (version 8.0): Omics data sets, evolutionary conservation,
regulatory phrases, cross-validated gold standards and more.// Nucleic Acids Research. 2013
Vol. 41 №D1: P. D203-D213.
2.Wingender E. TRANSFAC project as an example of framework technology that supports
the analysis of genomic regulation. // Brief Bioinform. 2008. Vol. 9. P.326-332.
3.Rigali S., Derouaux A., Giannotta F., Dusart J. Subdivision of the Helix-Turn-Helix GntR
Family of Bacterial Regulators in the FadR, HutC, MocR, and YtrA Subfamilies. // The journal
of Biol. Chem. 2002. Vol. 277. № 15. P. 12507-12515.
4.База данных http://www.ncbi.nlm.nih.gov/
5.Van Alten D.M.F., DiRusso C.C., Knudsen J., Wierenga R.K. Crystal structure of FadR, a
fatti acid-responsive transcription factor with a novel acyl coenzyme A-binding fold // The
EMBO Journal. 2000. Vol. 19. № 19. P. 5167-5177.
6.Kelley L.A., Sternberg M.J.E. Protein structure prediction on the web: a case study using
the Phyre server // Nature Protocols. 2009. Vol. 4. P. 363-371.
7.Mata-Gilsinger M., Ritzenthaler P., Blanco C. Characterization of the operator sites of the
exu regulon in Escherichia coli K-12 by operator-constitutive mutations and repressor titration
// Genetics. 1983. Vol.105 № 4. P. 829-42.
8.Suvorova I.A., Tutukina M.N., Ravcheev D.A., Rodionov D.A. et al. Comparative
genomics analysis of the hexuronate metabolism genes and their regulation in
gammaproteobacteria // J. of Bacteriology. 2011. Vol.193 № 15. P. 3956-3963.
9.Grainger D.C., Hurd D., Harrison M., Holdstock J., Busby S.J. Studies of the distribution
of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli
chromosome // Proc Natl Acad Sci U S A. 2005. Vol.6. 102 № 49. P.17693-17698.
10. Igarashi K., Ishihama A. Bipartite functional map of the E.coli RNA polymerase alpha
subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP //
Cell. 1991. Vol.65. P. 1015-1022.
11. Peekhaus N., Conway T. Positive and negative transcriptional regulation of the
Escherichia coli gluconate regulon gene gntT by GntR and cyclic AMP (cAMP)-cAMP
receptor protein complex // J. of Bacteriology. 1998. Vol.180. № 7. P. 1777-1785.
12. Shulami S., Gat O., Sonenshein A.L., Shoham Y. The glucuronic acid utilization
cluster from Bacillus stearothermophilus T-6// J.of Bacteriology. 1999. Vol. 181. № 12.
P 3695-3704.
13. Davis B.J. Disc electroforesis II. Method and application to human serum protein //
Ann. N.Y. Acad. Sci. 1994. Vol. 121. P. 404-427.
14. Tutukina M.N., Shavkunov K.S., Masulis I.S., Ozoline O.N. Antisense transcription
within the hns locus of Escherichia coli // Molecular biology. 2010. Vol. 44. № 3. P. 439-447.
15. Rodionov D.A., Mironov A.A., Rakhmaninova A.B., Gelfand M.S. Transcriptional
regulation of transport and utilization systems of hexuronides, hexuronates and hexonates in
gamma purple bacteria // Mol.Microbiol. 2000. Vol. 38. P. 673-683.
16. Рудаков О.Б., Востров И.А., Федоров С.В. и др. Спутник хроматографиста.
Методы жидкостной хроматографии. Воронеж: Водолей, 2004. 526 с
17. Peekhaus N., Conway T. What’s for dinner?: Entner-Doudoroff metabolism in
Escherichia coli. // J. Bacteriol. 1998. Vol. 180. P. 3495-3502.
García-Sotelo J.S. et al RegulonDB (version 8.0): Omics data sets, evolutionary conservation,
regulatory phrases, cross-validated gold standards and more.// Nucleic Acids Research. 2013
Vol. 41 №D1: P. D203-D213.
2.Wingender E. TRANSFAC project as an example of framework technology that supports
the analysis of genomic regulation. // Brief Bioinform. 2008. Vol. 9. P.326-332.
3.Rigali S., Derouaux A., Giannotta F., Dusart J. Subdivision of the Helix-Turn-Helix GntR
Family of Bacterial Regulators in the FadR, HutC, MocR, and YtrA Subfamilies. // The journal
of Biol. Chem. 2002. Vol. 277. № 15. P. 12507-12515.
4.База данных http://www.ncbi.nlm.nih.gov/
5.Van Alten D.M.F., DiRusso C.C., Knudsen J., Wierenga R.K. Crystal structure of FadR, a
fatti acid-responsive transcription factor with a novel acyl coenzyme A-binding fold // The
EMBO Journal. 2000. Vol. 19. № 19. P. 5167-5177.
6.Kelley L.A., Sternberg M.J.E. Protein structure prediction on the web: a case study using
the Phyre server // Nature Protocols. 2009. Vol. 4. P. 363-371.
7.Mata-Gilsinger M., Ritzenthaler P., Blanco C. Characterization of the operator sites of the
exu regulon in Escherichia coli K-12 by operator-constitutive mutations and repressor titration
// Genetics. 1983. Vol.105 № 4. P. 829-42.
8.Suvorova I.A., Tutukina M.N., Ravcheev D.A., Rodionov D.A. et al. Comparative
genomics analysis of the hexuronate metabolism genes and their regulation in
gammaproteobacteria // J. of Bacteriology. 2011. Vol.193 № 15. P. 3956-3963.
9.Grainger D.C., Hurd D., Harrison M., Holdstock J., Busby S.J. Studies of the distribution
of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli
chromosome // Proc Natl Acad Sci U S A. 2005. Vol.6. 102 № 49. P.17693-17698.
10. Igarashi K., Ishihama A. Bipartite functional map of the E.coli RNA polymerase alpha
subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP //
Cell. 1991. Vol.65. P. 1015-1022.
11. Peekhaus N., Conway T. Positive and negative transcriptional regulation of the
Escherichia coli gluconate regulon gene gntT by GntR and cyclic AMP (cAMP)-cAMP
receptor protein complex // J. of Bacteriology. 1998. Vol.180. № 7. P. 1777-1785.
12. Shulami S., Gat O., Sonenshein A.L., Shoham Y. The glucuronic acid utilization
cluster from Bacillus stearothermophilus T-6// J.of Bacteriology. 1999. Vol. 181. № 12.
P 3695-3704.
13. Davis B.J. Disc electroforesis II. Method and application to human serum protein //
Ann. N.Y. Acad. Sci. 1994. Vol. 121. P. 404-427.
14. Tutukina M.N., Shavkunov K.S., Masulis I.S., Ozoline O.N. Antisense transcription
within the hns locus of Escherichia coli // Molecular biology. 2010. Vol. 44. № 3. P. 439-447.
15. Rodionov D.A., Mironov A.A., Rakhmaninova A.B., Gelfand M.S. Transcriptional
regulation of transport and utilization systems of hexuronides, hexuronates and hexonates in
gamma purple bacteria // Mol.Microbiol. 2000. Vol. 38. P. 673-683.
16. Рудаков О.Б., Востров И.А., Федоров С.В. и др. Спутник хроматографиста.
Методы жидкостной хроматографии. Воронеж: Водолей, 2004. 526 с
17. Peekhaus N., Conway T. What’s for dinner?: Entner-Doudoroff metabolism in
Escherichia coli. // J. Bacteriol. 1998. Vol. 180. P. 3495-3502.
Published
2019-11-19
How to Cite
Potapova, A. V., Ozoline, O. N., & Tutukina, M. N. (2019). The development of effective overproduction and purification approaches for ExuR transcription factor of Escherichia coli using affine chromatography. Sorbtsionnye I Khromatograficheskie Protsessy, 14(3). Retrieved from https://journals.vsu.ru/sorpchrom/article/view/1500
Section
Статьи
