Hi experts, I need a lot EcoRV restrict enzyme for routine usage. I prepare to make by my own. As restricts are not normal protein, do I need special tricks to deal with the over-expressed enzyme for its activity toward coli genomic DNA and the recombinant plasmid? would the leakage of the T7lac promoter matter? Any established expression and purification protocol for type II restricts will be helpful. Thank you.
Hi Ziguo,
I think the trick will be to express the related EcoRV modification enzyme (methyltransferase) to prevent digestion of the genomic DNA. You may want a system where the methyltransferase is expressed constitutively (or expression is turned on well in advance of the restriction enzyme expression) to ensure complete protection of the genomic DNA. I can imagine that using something like LysS, etc. might be required to prevent leakage.
It might be worth approaching someone from NEB and asking for advice. 50 mg/day sounds a lot!
These links could be of interest:
https://www.neb.com/tools-and-resources/feature-articles/restriction-enzymes-at-neb-over-30-years-of-innovation
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310966/
Best wishes,
Roger
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