Restriction endonuclease from thermophilic bacterial species III. Isolation and characterization of BsiHKA I.

BsiHKA I is a type II restriction endonuclease from a Bacillus stearothermophilus strain isolated from soil according to (1). fisiHKA I in 11 culture (4.7 g wet cells) was purified by a DEAEsephacel column (30 ml bed volume). Enzyme eluted at about 0.3 M NaCl was dialysed against buffer A (1) and loaded onto a heparin column (8 ml bed volume). Enzyme eluted at 0.4—0.5 M NaCl was dialysed against buffer B (1) and loaded onto an FPLC Mono Q anion exchange column. Enzyme was eluted at 0.3-0.4 M NaCl. The purified enzyme was used to digest various DNA with known sequences (Fig. 1). The sizes and numbers of fragments produced are identical to those cleaved by mesophilic enzyme HgiAl which recognizes 5'G(AT)GC(AT)C3' (2). The cleavage site of BsiHKA I was determined according to (3) using pUC18 DNA as the template and a 17 mer oligonucleotide with sequence 5 'CAGCACTGACCC'1T1TG3' as the primer. The primer was annealed to position 359-375 of the denatured pUC18 DNA and was extended through the BsiHKA I site at position 444. Figure 2 shows that the cleavage product of reaction I comigrates with the band corresponding to nucleodde T in the sequence GAGCTC and reaction II comigrates with the band corresponding to the first G nucleodde. Therefore, BsiHKA I recognizes and cleaves 5'G(AT)GC(AT)!C3'. The optimal buffer for this enzyme was medium salt (4) and optimal reaction temperature 60°C.