Large-scale production of HIV-1 protease from Escherichia coli using selective extraction and membrane fractionation.
Human immunodeficiency virus type 1 (HIV-1) protease was expressed in Escherichia coli as a fusion protein with the N-terminal sequence of IGF-2. The protein accumulated in inclusion bodies as a 40:60 mixture of unprocessed fusion protein and processed protein. A simple purification procedure was developed that yielded 30-40 mg of active protease per liter of fermentation broth with a recovery of 30-40%. The purification process involved the selective extraction of HIV-1 protease from E. coli inclusion bodies with 50% acetic acid and fractional diafiltration to remove impurities and low-molecular-weight protease-related fragments. No chromatographic steps were employed, yet the HIV-1 protease produced by this procedure was greater than 95% pure by SDS-PAGE, reverse-phase HPLC, and N-terminal sequence analysis.
G.D. Searle and Co., Chesterfield, Missouri 63198, USA.
M E
ME
Gustafson
K D
KD
Junger
B A
BA
Foy
J A
JA
Baez
B F
BF
Bishop
S H
SH
Rangwala
M L
ML
Michener
R M
RM
Leimgruber
K A
KA
Houseman
R A
RA
Mueller
D000085:Acetates; D019342:Acetic Acid; D000595:Amino Acid Sequence; D005591:Chemical Fractionation; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D005822:Genetic Vectors; D016333:HIV Protease / Q000235:genetics* / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D000085:Acetates; D011993:Recombinant Fusion Proteins; D016333:HIV Protease; D019342:Acetic Acid