Molecular Characterization of HIV-2 (ROD) Protease Following PCR Cloning from Virus Infected H9 Cells
A 450 nucleotide sequence corresponding to the nucleotides 1931-2380 of the viral genome (8) was amplified by polymerase chain reaction (PCR) using template DNA prepared from HIV-2 (ROD) infected H9 cells. The sequence codes for HIV-2 protease and its N-terminal flanking peptide. An identical DNA sequence was obtained from three independent PCR amplifications, which differs from the published sequence of HIV-2 (ROD) in 7 nucleotides scattered throughout the region of the cloned DNA. The cloned DNA was expressed in E. coli cells and resulted in the synthesis of a correctly processed HIV-2 protease, which is enzymatically active. Therefore, none of the seven nucleotide changes, which resulted in two amino acid substitutions, affect the autoproteolytic or trans-cleaving activities of the HIV-2 protease.
KeywordsPhenol Agarose Coomassie Pepstatin Aspartyl
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