Affinity purification of HIV-1 and HIV-2 proteases from recombinant E. coli strains using pepstatin-agarose.
A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates. A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity. Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.
Rittenhouse
J
J
Pharmaceutical Products Division, Abbott Laboratories, IL 60064-3500.
Turon
M C
MC
Helfrich
R J
RJ
Albrecht
K S
KS
Weigl
D
D
Simmer
R L
RL
Mordini
F
F
Erickson
J
J
Kohlbrenner
W E
WE
D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli; D015684:Gene Products, pol / Q000235:genetics / Q000302:isolation & purification*; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology*; D015498:HIV-2 / Q000201:enzymology*; D008970:Molecular Weight; D010436:Pepstatins; D011994:Recombinant Proteins / Q000302:isolation & purification
D015684:Gene Products, pol; D010436:Pepstatins; D011994:Recombinant Proteins; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D016333:HIV Protease; C031375:pepstatin