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HIV-I protease

Cloning, Expression, and Purification

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Abstract

A new method for obtaining HIV-I protease was suggested. Fusion proteins composed of the N-terminal fragment of human γ-interferon and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed inEscherichia coli cells. The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein. Protein I was cleaved by enterokinase. The solubility of protein I was increased by treating with Nasulfite/Na-tetrathionate under denaturing conditions.

Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found. The hydrolysis products were separated by reversed-phase FPLC. The amount of tryptophan and cysteine residues in the enzyme obtained was estimated. The activity of HIV-I protease was determined using the chromogenic peptide AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of β galactosidase and a fragment ofgag proteins, including pl7-p24 processing site.

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Abbreviations

Tris:

tris(hydroxymethyl) aminomethane

MES:

2-(N-morpholino)ethanesulfonic acid

DTT:

dithiothreitol

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide gel electrophoresis

IPTG:

iso-propyl-β-thiogalactopyranoside

TFA:

trifluoroacetic acid

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117871, Moscow, Russia

    Natalya I. Dergousova, Alexander Yu. Amerik, Alla M. Volynskaya & Lev D. Rumsh

Authors
  1. Natalya I. Dergousova
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  2. Alexander Yu. Amerik
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  3. Alla M. Volynskaya
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  4. Lev D. Rumsh
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Correspondence to Natalya I. Dergousova.

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Dergousova, N.I., Amerik, A.Y., Volynskaya, A.M. et al. HIV-I protease. Appl Biochem Biotechnol 61, 97–107 (1996). https://doi.org/10.1007/BF02785692

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