Expression of an autoprocessing CAT-HIV-1 proteinase fusion protein: purification to homogeneity of the release 99 residue proteinase.
The 99 residue human immunodeficiency virus type 1 proteinase has been expressed in Escherichia coli as part of an autocleaving fusion protein. Expression of the fusion protein is toxic to the host cells, however yields of the released proteinase have been improved by optimising induction nad harvest times to increase culture biomass, and decrease degradation of the proteinase. Soluble proteinase was extracted from these cells by a simple and highly efficient three step process. N-terminal sequence analysis confirms that the enzyme preparation is highly pure and correctly autoprocessed. The proteinase cleaves peptide substrate IGCTLNFPISPIETV between F and P at pH 6.0 with a Km of 310 microM and a Kcat of 14s-1. The enzyme is sensitive to its ionic environment, showing stimulation of activity at high salt concentrations, and shows a pH optimising 5.5.
Montgomery
D S
DS
Department of Genetics, Glaxo Group Research Ltd, Greenford, Middlesex, United Kingdom.
Singh
O M
OM
Gray
N M
NM
Dykes
C W
CW
Weir
M P
MP
Hobden
A N
AN
D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D016333:HIV Protease / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D000727:Androgen-Binding Protein; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase; D016333:HIV Protease