Purification and biochemical characterization of FEN1: A structure specific endonuclease from Xiphophorus maculatus
Date
2005-05
Authors
Ruymgaart, Arnold P.
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Abstract
Flap endonuclease-1 (FEN1) is a structure specific 5' endo/exonuclease involved in DNA replication and repair. Presented herein are the cloning, gene structure, recombinant expression and characterization of flap endonuclease-1 (xiFENI) from Xiphophorus maculatus. The xiFENI gene structure was found to include 8 exons and 7 introns. The Xiphophorus FEN1 cDNA sequence harbored an open reading frame that encodes a 380 amino acid protein with a predicted mass of 43 kDa. The intact FEN1 cDNA was subcloned into a set of bacterial expression vectors (pET101-xiFEN1ct, pET100-xiFEN1nt and pET101-xiFENIwt) and recombinant xiFENI enzyme purified from E. coli cell extracts. The pET-xiFEN1 fusion translation products were tagged with a ~3 kDa vectorencoded carboxy (pET101 -xiFENIct) or amino (pET100-xiFEN1nt) terminal extension designed to facilitate protein recognition and purification. The xiFENI fusion proteins were purified and their amino acid sequences verified by Western blotting and tryptic peptide mass fingerprinting. The purified recombinant proteins were assessed for enzyme activity and specificity using several different oligonucleotide substrates. Results presented establish differences in kinetic parameters, substrate and product preference, and response to changes in temperature and metal ion cofactor for xiFENI activity compared to the human FEN1 protein.
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Keywords
endonucleases, DNA repair, recombinant proteins
Citation
Ruymgaart, A. P. (2005). Purification and biochemical characterization of FEN1: A structure specific endonuclease from Xiphophorus maculatus (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.