title: High-yield purification of HIV-1 proteinase expressed by a synthetic gene in Escherichia coli.
abstract: A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed. The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes. The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein. Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates. These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site. The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography. A high yield and a high specific activity were achieved.
authors.affiliation: Department of Virology, Karolinska Institute, Stockholm, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Goobar
authors.forename: U H
authors.initials: UH
authors.lastname: Danielson
authors.forename: P
authors.initials: P
authors.lastname: Brodin
authors.forename: T
authors.initials: T
authors.lastname: Grundström
authors.forename: B
authors.initials: B
authors.lastname: Oberg
authors.forename: E
authors.initials: E
authors.lastname: Norrby
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D005814:Genes, Viral; D016333:HIV Protease / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data
chemical_list: D004279:DNA, Viral; D016333:HIV Protease