General protocols of labeled protein production for NMR study
Expression and preparation of protein samples for NMR studies
Here I wish to give you general procedures of protein expression, purification and preparation for NMR studies in step by step. Most of these steps are very common for regular protein preparation by molecular biologist and biochemists. In some case you may need to spend some time to improve expression level, protein solubility and stability in the expression level and purification steps.
In general, proteins for NMR studies are expressed in bacterial expression systems (such as E. coli) as proteins are required to be isotopically labeled with 15N and/or 13C and/or 2H, and the labeled protein production in other systems such as mammalian and insect expression cells becomes very expensive. The isotope labeled proteins are expressed in E. coli using minimal medium (M9). I will give you details about M9 in the recipe section. Note: these are general procedures- please feel free to modify to your needs.
- Escherichia coli expression system: Since NMR studies require large quantities of stable isotope labeled proteins; E. coli expression system is the best and cheapest way to produce them. There are number of vectors available for example: pET, pGEX, pMal, pTTB11. It is common to add enzymatically cleavable tag/s (6His, GST, MBP etc.) for affinity purification. It is always good to check expression level in small scale with an appropriate cell line, such as BL21 (DE3), BL21(DE3) pLYS etc. If protein expression level is low and has low solubility, then one need to explore systematic optimization: changing construct length (bioinformatics), cell lines, induction time & temperature and buffer optimization etc. Details can be found elsewhere.
- Protein production: Once expression level is optimized in LB medium, then you should check expression level in minimal medium in small scale before you start a large scale labeled protein production. Here are general steps for labeled protein production:
- Inoculate a single colony from a freshly transformed plate into 5 ml of LB medium
- Grow culture till OD ~0.6
- Take 50 ul to 100 ml minimal medium (see M9) and grow overnight
- Add 50 ml to 1 liter minimal medium
- Grow cells and induce with IPTG at OD ~0.6 (or as appropriate)
- Induce it for 4-6 hours at 37 OC or overnight at 20 OC (sometimes low temperature induction helps with protein stability and solubility).
If you need to label your protein with 2H, please get in touch with me: firstname.lastname@example.org
- Purification: This is a key and probably the most time consuming step. I recommend that you should optimize each purification steps with unlabeled protein (preferably produced in LB media) and then attempt with label protein purification. I also advise you to run a 1H 1D NMR experiment on an unlabeled protein purified from LB or unlabeled M9 media to check if purified protein is folded. Then I encourage you to produce and purify only 15N labeled only protein (since 15N labeling is cheaper compared with 13C or both 13C & 15N labeling). This protein will be used to check protein stability at room temperature (RT) by collecting several 2D heteronuclear experiments such as HSQC at 4-5 days interval. This is important step because if protein is unstable you may have to improve buffer condition in which protein stays stable for several days at RT as each 3D NMR experiment requires one and half to three days. Here are general steps for protein purification with some tips:
- Lyse cells in lysis buffer: A typical lysis buffer contains – 20-50 mM tris @ pH 7.5, 100 -150 mM NaCl and protease inhibitors. Add reducing agent (DTT/b-mercaptoethanol/TCEP) and/or 0.5-1% glycerol if required.
- Sonication is widely used to lyse cells but I recommend to use French press. Sonication may sometime introduce protein unfolding and aggregation.
- Centrifuge at 15000 rpm (fixed rotors) for 30 minutes to separate cell debris and supernatant.
- If protein is tagged then use affinity column first to separate your protein of interest from other un-wanted bacterial proteins. If it is his-tagged, use Ni-NTA or Talon beads (Qiagen or Clonetech). Follow their protocol for binding and eluting proteins.
- In most case proteins are not enough pure to do NMR, you may have to pass through sizing (also known as gel filtration) column to further purify the protein. Also if you want the tag to be removed from your protein then you can treat your protein with appropriate enzyme then do sizing column. One can also protein remove tag from affinity column by treating bounds beads with enzyme and then elute tag-less protein prior to sizing column.
- Check your protein purity at each step of protein purification by SDSPAGE and at the last step I also encourage you to do mass spec as well to confirm mass. If further purification required then consider using ion-exchange column.
- Concentrate your protein to a desire concentration for NMR studies. If buffer at the last step is different from your NMR buffer, I suggest you to dialyze first in your NMR buffer and then concentrate.
- Concentration: 500 uM or higher concentration are desirable for structural studies. You can get away with 200 to 300 uM protein if you have access to cryoprobe and higher magnetic field NMR instruments. Usually to check protein stability and folding, 50 uM protein is enough for HSQC experiment. Please also pay attention to pH of NMR sample. You should keep your protein at least one pH unit away from its PI and try to keep protein happy in NMR buffer. Lower pH between 4 and 6 is preferable for NMR studies.
- Buffer: Most buffers are suitable for NMR studies (phosphate, tris, MES, bis-tris etc).
- Recipe for M9 medium per liter
- 6 g Na2HPO4
- 3 g KH2PO4
- 0.5 g NaCl
- 1 g NH4Cl (for 15N source)
- 5 g glucose (for 13C-labeled, use 2 g)
- 1 ml of 1 M MgSO4
- 200 ul of 1 M CaCl2
- 1 ml of Thiamine (40 mg/ml stock)
- 10 ml trace element
- Trace Elements per liter (pH 7.0)
- 0.5 g FeCl3 anhydrous or 0.8 g FeCl3.6H2O
- 0.05 g ZnCl2
- 0.01 g CuCl2
- 0.01 g CoCl2.6H2O
- 0.01 g H3BO3
- 1.6 g MnCl2.6H2O or 1.35 g MnCl2.4H2O
Preparation of M9:
Weigh appropriate amount of Na2HPO4, KH2PO4, NaCl, NH4Cl, glucose and add to 950 ml of H2O one by one while stirring solution with a magnetic bar. Add 200 ul of 1M CaCl2 stock solution drop by drop (care must be taken as Ca3(PO4)2 will precipitate) to the solution. Then add 1 ml of 1M MgSO4 solution, 1 ml of Thiamine (40 mg/ml) and 10 ml of trace elements. Then filter sterile the solution.
Preparation of Trace element:
Add ingredients one by one to 900 ml of water while stirring with a magnetic bar. Adjust pH to 7.0 and final solution should brown and then filter sterile it.
Here is a link for further reading:
GE Healthcare Life Science provides details about protein expression and purification protocols.