Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus

摘要

The DNA polymerase gene of Thermococcus marinus (Tma) contains an inteininserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acidresidue. The LAGLIDADG motif, often found in site-specific DNA endonucleases,was detected within the amino acid sequence of the intein. The intein endonu-clease, denoted as PI-Tma, was purified as a naturally spliced product from theexpression of the complete DNA polymerase gene in Escherichia coli. PI-Tmacleaved intein-less DNA sequences, leaving four-base-long, 3'-hydroxyl overhangswith 5'-phosphate. Nonpalindromic recognition sequences 19 bp long were alsoidentified using partially complementary oligonucleotide pair sequences insertedinto the plasmid pET-22b( + ). Cleavage by PI-Tma was optimal when present in50 mM glycine–NaOH (pH 10.5), 150 mM KC1 and 12 mM MgCl_2 at 70 ℃.