HIV-1 INTEGRASE EXPRESSED IN ESCHERICHIA-COLI FROM A SYNTHETIC GENE

Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from t he gag-pol precursor by the HIV1 protease. The resulting 32-kDa protei n is used by the infecting virus to insert a linear, double-stranded D NA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to faci litate mutagenesis, we have designed and synthesized a gene encoding t he integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restrictio n sites were incorporated throughout the gene. A 905-bp cassette conta ining a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two prime rs (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and m olecular mass, and displays the DNA processing, DNA joining and disint egration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the grou ndwork for structure-function studies of HIV1 integrase.