Comparison of HIV-1 protease expression in different fusion forms

Biochem Mol Biol Int. 1995 Jun;36(2):411-9.

Abstract

Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters*
  • Amino Acid Sequence
  • Bacteriophage T7 / genetics
  • Base Sequence
  • Blotting, Western
  • Carrier Proteins / genetics
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Escherichia coli Proteins*
  • Gene Expression* / genetics
  • Genes, Viral
  • Genetic Vectors / genetics
  • Glutathione Transferase / genetics
  • HIV Protease / biosynthesis
  • HIV Protease / chemistry
  • HIV Protease / genetics*
  • HIV Protease / metabolism
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Maltose-Binding Proteins
  • Molecular Sequence Data
  • Monosaccharide Transport Proteins*
  • Protein Processing, Post-Translational / genetics
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism

Substances

  • ATP-Binding Cassette Transporters
  • Carrier Proteins
  • Escherichia coli Proteins
  • Maltose-Binding Proteins
  • Monosaccharide Transport Proteins
  • Recombinant Fusion Proteins
  • maltose transport system, E coli
  • Glutathione Transferase
  • HIV Protease