We grew in E. Coli BL21DE3 at temperatures of 37 and 18C. Label (unlabel) incorporation for 13C was excellent for ILFY aa's, when the aa's are added about 1 hr before induction. We found a lot of scrambling for 15N with the longer induction times at 18C. We split the culture 8:2 or 7:3 and dissolved the aa's in the small part, then added them when the large part of the culture reached the induction OD, thus diluting the culture to require about another hour of growth. Certain aa's will not label well no matter what in E. Coli, such as glutamic acid and serine. Which aa's are you trying to unlabel? what are the growth condition, and organism?

Agree with what LA said. You are probably using EColi. There are only a couple of aa you can label selectively in BL21(DE3) (K and A) The rest will all scramble to some extent. You can try auxotroph cells for the particular aa you want to label. You will get less scrambling. As LA mentioned S is a difficult one and it mostly scrambles to G. You can suppress some of this by adding G to the media before induction.

Thanks Loren and Tamjeed.

Loren: After adding aa in the smaller part of 8:2 ratio culture, do you used to grow both the part to reach the OD 0.8 and then mixed or, just the larger part(where aa is not added) you used to grow to reach the OD 0.8 and then the smaller part(where aa is added) you mixed without growing the latter after adding aa?

I used to grow at 37C and induction at 25C . I am trying to unlabel Asp,Ile and Leu separately.

Tamjeed: The problem is with selective unlabeling of aa, I am not doing selective labeling . R,N,K,H does not cross metabolises to other aa during the process, but other aa like D,I,L shows strong scrambling effect. My problem is with that. And by using auxotroph strains or, cell-free synthesis or, in vitro expression systems which lack these enzymes for cross metabolism to other aa the effect can be minimized. But, the question is how it can be minimized with this E.coli. systems by doing some changes like Loren has mentioned one or, like that some changes which can minimize this scrambling.

Hi Somnath,

I'll try to clarify: If you grow 1 L, split off 700 mL for the growth and grow up to your induction OD. Use the other 300 mL to dissolve the AA's, which can take several hours. Add the 300 mL aa mix to the 700 mL culture, so now the OD will be at 70% of the induction OD. Then let grow for 1 hr or so and the culture should be back at the correct OD for induction. Now induce as you normally do.

You can try to keep the induction times short to minimize scrambling. If you are trying to unlabel 15N and low temperature, It may not be successful due to transaminase. In our hands, the 13C unlabels well, but 15N is almost fully exchanged due to transaminase activity after 18 hours at 18 C. If you are trying to label 13C, you should have no problem with I and L, even for fairly long induction times (>18 hrs at 18C is fine). I would avoid D because it will feed right into the TCA cycle. The following publication may be helpful for 13C labeling:

J Biomol NMR (2009) 43:111–119 DOI 10.1007/s10858-008-9294-7

An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein

Isabel Ayala Æ Remy Sounier Æ Nathalie Use ́ Æ Pierre Gans Æ Je ́roˆme Boisbouvier

Thank you very much Loren. I will try this.

Hi all - interesting and practical discussion here, I'd like to join in with a couple of points.

1). Somnath: I completely agree with prior posters on two key points: 1). unlabeling 13C for I/L should work well; 15N can be problematic. 2). Asp will be challenging. With a bit more information about your application (e.g. do you need to completely unlabel all of the aa, or just sidechain? can you confirm 13C is what you need?), I think there are some good resources that you can be pointed to.

2). Loren: Interesting idea to handle the poor solubility of certain aa, but I've got a slight concern about the potential for setting up two distinct pools of bacteria when you're inducing: one which has seen the aa for only 1 hour, and another which has seen the aa for 1hr + preceding incubation time. As such, one could potentially produce protein in a sample with two different labeling efficiencies but overlapping signals in many experiments. For many experiments, this wouldn't have an effect; for others, particularly those where you're looking to quantitate (isotope-filtered NOESY?), one might interpret intensity differences incorrectly as a result.

This concern might simply be academic --- aa uptake/incorporation rates might be fast compared to various incubation times, quantitation might not be needed, etc. --- but I think I'd err on the safe side by growing that initial culture as simply 700mL, reserving the other 300mL to incubate the aa without bacteria present. Then add as you suggest.

Hi Kevin. We're both talking about growing the same way. I wrote 'culture' when I should have written 'media' in my first post. So, yes, the 300 mL fraction just has media+aa's when we do this kind of growth.

Excellent - we're all on the same page here, thanks for the clarification.

Kevin: First of all thanks for your valuable suggestion. I need completely unlabeled aa, not just side chains. Actually, I am trying first this with just 15N labeling, as Loren has mentioned and you have rectified the protocol part, I will check with that for sure. I don't have any plan for 13C labeling at the present moment.

Dear all.... very interesting discussion. As we already know that it is straight forward to get assignments for three amino acids: (S,T and G) just based on their Cb chemical shifts so no need to unlabel them.

It is easy to quantify the percentage of scrambling just by comparing the peak intensities (15N-HSQC) of selectively unlabelled and uniformly labelled spectra. But this is possible when the spectrum with well resolvedresonances and or in case of small proteins.So it is not a good idea to work with aa which undergo metabolic scrambling. I have got excellent results with a large protein (46 kDa) where I did prepared R,K,H and ILV (together) unlabelled samples. So I would prefer to unlabel only R,K,H and ILV together (as they scramble to each other).

Thus residue specific assignments for S,T,G,R,K,H and ILV are straight forward. Further ILV residues can be differentiate based on their Cb chemical shifts together with Cb chemical shifts of their sequential residues in HNCACB.

I hope this information will help you.

Good Luck

Krishna.