I want to over express a protein in the host strain (gram positive). What will be strategy for vector selection and transformation? Can I use an inducible promoter or not?
The obvious advantage of an inducible promoter is low (if any) expression without the inducing agent, so there's your control already. Moreover, should your protein of interest turn toxic or disadvantageous, playing with the expression levels by adjusting the inducing agent can get you right once more.
DO NOT FORGET to tag your protein for Western detection already while cloning!!! (His-tag, FLAG, you name it...).
I had good experience with the traditional GalA promoter (E.coli; pBad).
Hope this helps.
Thanks Marcin,
But my majour question is vector selection. which vector will be suitable for transformation in my strain (bacillus) that is wild type.
Word of warning: be careful about your tag selection. I've heard that people are starting to see that certain tags can change a protein and it's interactions significantly (add significant charge, change hydrophobicity, etc...) so be thoughtful of this. Also, tags couldinteract with native proteins differently in your strain than in E coli.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins