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Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product

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Wolber,  Vera
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Rensland,  Hans
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Brandmeier,  Birgit
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Kalbitzer,  Hans Robert
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Wittinghofer,  Alfred
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Wolber, V., Rensland, H., Brandmeier, B., Sagemann, M., Hoffmann, R., Kalbitzer, H. R., et al. (1992). Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product. European Journal of Biochemistry, 205(3), 1115-1121. doi:10.1111/j.1432-1033.1992.tb16880.x.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0019-AB78-C
Abstract
The human immunodeficiency virus 1 (HIV-1) nef gene encoded by the HIV-1 isolate lymphadenopathy-associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter. The protein is found mainly in the soluble part of the bacterial lysate; a simple two-column purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents. Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a beta-pleated sheet content of 31%. GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well-characterized guanine-nucleotide-binding (GNB) protein. Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass less than 5 x 10(3) M-1). An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 x 10(3) M-1. Nef can be autophosphorylated by ATP; under the experimental conditions used, 1-2% of the protein become phosphorylated. Correspondingly, our Nef preparation shows a low, but significant, ATPase activity. In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded.