Complete 1H nuclear magnetic resonance assignments and structural characterization of a fusion protein of the alpha-amylase inhibitor tendamistat with the activation domain of the human immunodeficiency virus type 1 Tat protein.

Complete sequence-specific assignments of the 1H-NMR spectrum of a fusion protein of the alpha-amylase inhibitor tendamistat from Streptomyces tendae and the activation domain of Tat from human immunodeficiency virus type 1 (HIV-1) was obtained by homonuclear two-dimensional NMR methods. The protein behaves as expected for an ideal fusion protein: the flexible linker allows an almost completely decoupled motion of the subunits of the protein and the two subunits show almost no mutual interaction. In the tendamistat part, small structural distortions due to exchange of the carboxy-terminal leucine propagate mainly via the hydrogen bonds of the beta-sheet and the disulfide bond.

A possible regulation of negative factor (Nef) activity of human immunodeficiency virus type 1 by the viral protease.

Negative factor (Nef) protein from human immunodeficiency virus type 1 (HIV-1) is cleaved into two well-defined domains by the HIV-1-encoded protease. The cleavage site is located between Trp57 and Leu58 and is well conserved. The two domains are stable in the presence of protease for more than 48 h.

A universal expression-purification system based on the coiled-coil interaction of myosin heavy chain.

We have constructed a series of Escherichia coli expression vectors that produce high yields of fusion proteins containing the C-terminal fragment of light meromyosin (LMM) from rabbit fast skeletal muscle. The fusion proteins retain the ability of LMM to form polymers in low salt and to be soluble in high salt. Thus they can be easily purified from bacterial extracts with a high salt-low salt extraction procedure and still retain their biochemical properties.

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

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%.

Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera.

The prevalence of antibodies against HIV-1 regulatory proteins in sera of HIV-infected patients from different stages of disease was investigated. HIV-1 vif, tat, and nef genes were cloned in procaryotic vectors and were expressed as MS-2 fusion proteins (vif and nef) or as a non-fusion protein (tat). These recombinant proteins were employed in immunoblot experiments.