HIV1 integrase expressed in Escherichia coli from a synthetic gene

Tod P. Holler, Susan K. Foltin, Qizhuang Ye, Donald J. Hupe

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA 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 facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing 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 primers (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 molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.

Original languageEnglish (US)
Pages (from-to)323-328
Number of pages6
JournalGene
Volume136
Issue number1-2
DOIs
StatePublished - Dec 22 1993
Externally publishedYes

Fingerprint

Synthetic Genes
Integrases
HIV-1
Escherichia coli
DNA
Codon
Nucleotides
Genes
Initiator Codon
Oligodeoxyribonucleotides
Viral RNA
Ribosomes
Mutagenesis
Reverse Transcription
Proteins
Peptide Hydrolases
Clone Cells
Binding Sites
Genome
Viruses

Keywords

  • AIDS
  • gene synthesis
  • mutagenesis
  • oligodeoxyribonucleotide
  • Recombinant DNA
  • retro viral protein, polymerase chain reaction

ASJC Scopus subject areas

  • Genetics

Cite this

HIV1 integrase expressed in Escherichia coli from a synthetic gene. / Holler, Tod P.; Foltin, Susan K.; Ye, Qizhuang; Hupe, Donald J.

In: Gene, Vol. 136, No. 1-2, 22.12.1993, p. 323-328.

Research output: Contribution to journalArticle

Holler, Tod P. ; Foltin, Susan K. ; Ye, Qizhuang ; Hupe, Donald J. / HIV1 integrase expressed in Escherichia coli from a synthetic gene. In: Gene. 1993 ; Vol. 136, No. 1-2. pp. 323-328.
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