Abstract
The DNA polymerase gene of Thermococcus marinus (Tma) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI-Tma, was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli. PI-Tma cleaved intein-less DNA sequences, leaving four-base-long, 3′-hydroxyl overhangs with 5′-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI-Tma was optimal when present in 50 mM glycine-NaOH (pH 10.5), 150 mM KCl and 12 mM MgCl2 at 70 °C.
Original language | English (US) |
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Pages (from-to) | 180-188 |
Number of pages | 9 |
Journal | FEMS Microbiology Letters |
Volume | 297 |
Issue number | 2 |
DOIs | |
State | Published - Aug 2009 |
Externally published | Yes |
Keywords
- Endonuclease
- Intein
- Thermococcus marinus