I have a small protein domain that expresses well in LB but may be membrane associated and so toxic to E coli when I grow it in minimal media. I want to be able to 15N label my protein. I have tried growing in LB and expressing in minimal media but nothing either. Has anyone got any protocols or suggestions where they have managed to get expression with otherwise I was impossible?
Thanks
Chris
Hi Chris,
Have you considered using IsoGro for your expression? You can either use it as a complete growth medium at the concentration suggested by Sigma or we sometimes use it as a supplement to minimal media at 10 % of the concentration (if memory serves). I think you can get it unlabelled for tests and the price is reasonable for both 15N and 13C labelling especially if you ask a Sigma rep for a quote.
Message me if you'd like any further advice.
Best of luck.
Andy
IsoGro is very good for minimal media expression. I wanted to ask how you determined that the protein is toxic. After induction do you see the OD of your culture decrease? If thats the case you may try to induce it later and harvest sooner. There are some cell lines like C41/43 that do better with toxic proteins. Also the Lemo cells from NEB.
Thanks Andrew, I have ordered a trial of the ISOGRO so I'll see how I go with that. Tamjeed, I am already using C41 cells but a good idea non the less. I have tried different growth conditions so I will see how i get on with the ISOGRO. Thank you both for your help!
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