Overproduction and characterization of the StsI restriction endonuclease.
The StsI restriction endonuclease (R-StsI), a class-IIS restriction endonuclease, found in Streptococcus sanguis 54, is a heteroschizomer of R-FokI, which recognizes 5'-GGATG-3'. To overproduce R-StsI in Escherichia coli, the coding region of R-StsI was joined to the tac promoter of an expression vector, pKK223-3. By introduction of the plasmid into E. coli UT481 cells expressing the fokIM gene, R-StsI activity was overproduced, from which R-StsI was purified homogeneously. We compared the properties of R-StsI with those of R-FokI. The optimum reaction conditions for R-StsI were quite different fron those for R-FokI. R-StsI is an acidic protein (pI 6.3). Anti-R-StsI serum did not cross-react with R-FokI, indicating three-dimensional structural dissimilarity. The domain structure of R-StsI was elucidated by digestion with trypsin. In the presence of substrate DNA, R-StsI was digested to yield 45-kDa N-terminal and 23-kDa C-terminal fragments. The amino-acid sequences around the trypsin cleavage sites of R-StsI and R-FokI were quite homologous.
Kita
K
K
Department of Biotechnology, Tottori University, Japan. kita@bio.tottori-u.ac.jp
Suisha
M
M
Shintoh
M
M
Yanase
H
H
Kato
N
N
D000595:Amino Acid Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000302:isolation & purification*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins; D013298:Streptococcus sanguis / Q000201:enzymology*
D011994:Recombinant Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific