Comparison of HIV-1 protease expression in different fusion forms.
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)
Wan
M
M
Department of Biochemistry, Faculty of Medicine, National University of Singapore.
Takagi
M
M
Loh
B N
BN
Imanaka
T
T
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D016333:HIV Protease / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D005982:Glutathione Transferase; D016333:HIV Protease