Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae.
Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands. To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast. The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal amino acid codons. Inclusion of a polyhistidine tag facilitates purification of recombinant IN by metal chelate chromatography. Recombinant Ty1 IN is active in an in vitro assay with short double-stranded oligonucleotide substrates and has biochemical properties similar to those observed with Ty1 virus-like particles. The full-length Ty1 IN produced in yeast should be useful for further biochemical, genetic, and structural analyses of Ty1 integration and for comparative analyses with retroviral IN proteins.
Moore
S P
SP
National Cancer Institute-Frederick Cancer Research and Development Center Advanced BioScience Laboratories-Basic Research Program, Frederick, MD 21702-1201.
Garfinkel
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D019426:Integrases; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D016662:Virus Integration
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases