High-level expression and purification of mature HIV-1 protease in Escherichia coli under control of the araBAD promoter.
A 1.3-kb segment of Escherichia coli DNA containing the regulatory gene, araC, and the promoter of the araBAD operon was amplified by the polymerase chain reaction (PCR) and cloned into pUC18, resulting in plasmid pKB130 that produced the alpha fragment of beta-galactosidase upon addition of L-arabinose (L-ara). A synthetic gene for human immunodeficiency virus (HIV)-1 preprotease was placed downstream of the ara-BAD promoter in pKB130 to create a translational fusion inducible by addition of L-ara. The fusion protein correctly autoprocessed in vivo to yield a mature 99-amino-acid HIV-1 protease, which was found predominantly in inclusion bodies. This material could be refolded to an active form, which was purified to homogeneity. A small fraction of the protease was expressed in vivo as a soluble active form, which allowed the monitoring of expression during fermentation by a rapid and simple whole cell assay employing an HIV-1 protease-specific fluorogenic substrate.
Corporate Molecular Biology, Abbott Laboratories, IL 60064.
A
A
Taylor
D P
DP
Brown
S
S
Kadam
M
M
Maus
W E
WE
Kohlbrenner
D
D
Weigl
M C
MC
Turon
L
L
Katz
D000595:Amino Acid Sequence; D001089:Arabinose / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D016333:HIV Protease / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping
D011993:Recombinant Fusion Proteins; D001089:Arabinose; D016333:HIV Protease