Abstract
A simple and efficient method for expression in Escherichia coli cells and purification of a recombinant matrix protein, p17, of human immunodeficiency type I virus has been described. HIV-1 subtype A DNA sequence encoding p17 was obtained by amplification of the viral gag gene segment and cloned into an expression vector under the control of T7Lac promoter. The conditions for cell growth and induction of p17 synthesis by lactose and its further purification by metal chelate chromatography were optimized. p17 preparations with 97% purity were obtained; the yield of the protein of 28 mg per 1l of culture was achieved. The obtained protein was capable of binding antibodies from blood serum of a HIV-infected patient during immunoblotting.
Abbreviations
- HAART:
-
highly active anti-retroviral therapy
- IL:
-
2-interleukin-2
- IFN-γ:
-
interferon γ
- IPTG:
-
isopropyl-β-D-thiogalactopyranoside
- OD:
-
optical density
- PAGE:
-
polyacrylamide gel electrophoresis
- PCR:
-
polymerase chain reaction
- LB medium:
-
Luria-Bertani medium
- TNF-α:
-
tumor necrosis factor α
- PBS:
-
phosphate buffered saline
References
De Francesco, I.A., Baronio, M., Fiorentini, S., Signorini, C., Bonfanti, C., Poiesi, C., Popovic, M., Grassi, M., Garrafa, E., Bozzo, L., Lewis, G.K., Licenziati, S., Gallo, R.C., and Caruso, A., Proc. Natl. Acad. Sci. USA, 2002, pp. 9972–9977.
Vitale, M., Caruso, A., Francesco, M.A., and Rodella, L., Bozzo, L., Garrafa, E., Grassi, M., Gobbi, G., Cacchioli, A., and Fiorentini S., Br. J. Haematol., 2003. vol. 4. pp. 337–343.
De Francesco, M.A., Poiesi, C., Ricotta, D., and Manca, N., Virus Res., 2006, vol. 118, pp. 170–177.
Marini, E., Tiberio, L., Caracciolo, S., Tosti, G., Guzman, C.A., Schiaffonati, L., Fiorentini, S., and Caruso, A., Cell Microbiol., 2008, vol. 10. pp. 655–666.
Pulliam, L., Sun, B., ana Rempel, H., J. Neuroimmunol., 2004, vol. 157, pp. 93–98.
Fiorentini, S.E. Riboldi, F. Facchetti, M. Avolio, M. Fabbri, Tosti, G., Becker, P.D., Guzman, C.A., Sozzani, S., and Caruso, A., Proc. Natl. Acad. Sci. USA, 2008, vol. 105, pp. 3867–3872.
De Francesco, M.A., Caruso, A., Fallacara, F., Canaris, A.D., Dima, F., Poiesi, C., Licenziati, S., Corulli, M., Martinelli, F., Fiorentini, S., and Turano, A., AIDS, 1998, vol. 12, pp. 245–252.
Popovic, M., Tenner-Racz, K., Pelser, C., Stellbrink, H.J., Van Lunzen, J., Lewis, G., Kalyanaraman, V.S., Gallo, R.C., and Racz, P., Proc. Natl. Acad. Sci. USA, 2005, vol. 102, pp. 14807–14812.
Studier, F.W., Protein Expr. Purif., 2005, vol. 41, pp. 207–234.
Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.
Bradford, M.M., Anal. Biochem., 1976, vol. 72, pp. 248–254.
Baneyx, F., Curr. Opin. Biotechnol., 1999, vol. 10, pp. 411–421.
Grossman, T.N., Kawasaki, E.S., Punreddy S.R., and Osburne, M.S., Gene, 1998, vol. 209, pp. 95–103.
Burnette, B., Kahn, R., Glover, C.J., and Felsted R.L., Protein Expr. Purif., 1992, vol. 3, pp. 395–402.
Massiah, M.A., Starich, M.R., Paschall, C., Summers, M.F., Christensen, A.M., and Sundquist, W.I., J. Mol. Biol., 1994, vol. 244, no. 2, pp. 198–223.
Matthews, S., Barlow, P., Boyd, J., Barton, G., Russell, R., Mills, H., Cunningham, M., Meyers, N., Burns, N., Clark, N., Kingsman, S., Kingsman, A., and Campbell, I., Nature, 1994, vol. 370, pp. 666–668.
Saito, A., Morimoto, M., Ohara, T., Takamizawa, A., Nakata, A., and Shinagawa, H., Microbiol. Immunol., 1995, vol. 39, pp. 473–483.
Ishikawa, S., Hashinaka, K., Hashida, S., Oka, S., and Ishikawa, E., J. Clin. Lab. Anal., 1998, vol. 12, pp. 343–350.
Gupta, S., Arora, K., Gupta, A., and Chaudhary, V.K., Protein Expr. Purif., 2001, vol. 21, pp. 378–385.
Author information
Authors and Affiliations
Corresponding author
Additional information
Original Russian Text © R.I. Al-Shekhadat, I.V. Dukhovlinov, A.I. Kobatov, N.A. Klimov, A.P. Kozlov, 2010, published in Biotekhnologiya, 2010, No. 6, pp. 19–26.
Rights and permissions
About this article
Cite this article
Al-Shekhadat, R.I., Dukhovlinov, I.V., Kobatov, A.I. et al. Obtaining of the p17 recombinant protein of human immunodeficiency subtype A virus. Appl Biochem Microbiol 47, 730–736 (2011). https://doi.org/10.1134/S0003683811080023
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0003683811080023
Keywords
- p17 protein
- human immunodeficiency virus
- HIV-1
- subtype A
- Gag