LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity
Апстракт
Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13 degrees C and 37 degrees C) and was stable after storage at -20 degrees C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts anothe...r alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems.
Извор:
Biomed Research International, 2018, 2018Издавач:
- Hindawi Ltd, London
Финансирање / пројекти:
- Изучавање гена и молекуларних механизама у основи пробиотичке активности бактерија млечне киселине изолованих са подручја западног Балкана (RS-MESTD-Basic Research (BR or ON)-173019)
DOI: 10.1155/2018/5657085
ISSN: 2314-6133
PubMed: 29789800
WoS: 000423797000001
Scopus: 2-s2.0-85042145835
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Miljković, Marija AU - Malešević, Milka AU - Filipić, Brankica AU - Vukotić, Goran AU - Kojić, Milan PY - 2018 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/1156 AB - Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13 degrees C and 37 degrees C) and was stable after storage at -20 degrees C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems. PB - Hindawi Ltd, London T2 - Biomed Research International T1 - LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity VL - 2018 DO - 10.1155/2018/5657085 ER -
@article{ author = "Miljković, Marija and Malešević, Milka and Filipić, Brankica and Vukotić, Goran and Kojić, Milan", year = "2018", abstract = "Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13 degrees C and 37 degrees C) and was stable after storage at -20 degrees C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems.", publisher = "Hindawi Ltd, London", journal = "Biomed Research International", title = "LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity", volume = "2018", doi = "10.1155/2018/5657085" }
Miljković, M., Malešević, M., Filipić, B., Vukotić, G.,& Kojić, M.. (2018). LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity. in Biomed Research International Hindawi Ltd, London., 2018. https://doi.org/10.1155/2018/5657085
Miljković M, Malešević M, Filipić B, Vukotić G, Kojić M. LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity. in Biomed Research International. 2018;2018. doi:10.1155/2018/5657085 .
Miljković, Marija, Malešević, Milka, Filipić, Brankica, Vukotić, Goran, Kojić, Milan, "LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity" in Biomed Research International, 2018 (2018), https://doi.org/10.1155/2018/5657085 . .