Document 0922
 DOCN  M9440922
 TI    HIV1 integrase expressed in Escherichia coli from a synthetic gene.
 DT    9404
 AU    Holler TP; Foltin SK; Ye QZ; Hupe DJ; Department of Biochemistry,
       Parke-Davis Pharmaceutical Research,; Division of Warner-Lambert
       Company, Ann Arbor, MI 48105.
 SO    Gene. 1993 Dec 22;136(1-2):323-8. Unique Identifier : AIDSLINE
       GENBANK/L21188
 AB    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.
 DE    Amino Acid Sequence  Base Sequence  Cloning, Molecular  DNA
       Nucleotidyltransferases/BIOSYNTHESIS/*GENETICS/ISOLATION &
       PURIF/METABOLISM  DNA, Recombinant  Electrophoresis, Polyacrylamide Gel
       Escherichia coli  *Genes, Synthetic  HIV-1/*ENZYMOLOGY  Molecular
       Sequence Data  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).