Large-scale production of HIV-1 protease from Escherichia coli using selective extraction and membrane fractionation.

Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.

Large-scale production of HIV-1 protease from Escherichia coli using selective extraction and membrane fractionation.

Protein Expr Purif. 1995 Aug;6(4):512-8. Unique Identifier : AIDSLINE MED/96136639
Gustafson ME; Junger KD; Foy BA; Baez JA; Bishop BF; Rangwala SH; Michener ML; Leimgruber RM; Houseman KA; Mueller RA; et al; G.D. Searle and Co., Chesterfield, Missouri 63198, USA.

Abstract: 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.

Keywords: Acetic Acids Amino Acid Sequence Escherichia coli/*GENETICS Fermentation Fractionation Genetic Vectors HIV Protease/*GENETICS/*ISOLATION & PURIF Inclusion Bodies/ENZYMOLOGY Intracellular Membranes/ENZYMOLOGY Molecular Sequence Data Plasmids Recombinant Fusion Proteins/GENETICS/ISOLATION & PURIF JOURNAL ARTICLE $KWDgeneticsfermentationfractionationgeneticvectorshivprotease/$
960430
M9640813

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