Samples for NMR
For each protein, we usually make the following samples:
- [U-15N, 13C]-labeled sample, for resonance assignment and NOE interpretation.
A U-15N and fractional 5%-13C-labeled sample for stereospecific assignment of Val and Leu isopropyl methyl groups based on the method of Neri et. al. (Biochemistry 28, 7510-7516,1989) that we call the NC5 sample. This sample is prepared using 5% U[1H,13C]-D-glucose and 95% unlabelled glucose in the E. coli minimal growth media.
The NC5 sample is not [U-5%-13C,U-15N] since it is not uniformly 5% -13C labeled. The incorporation of 13C in the methyl groups is biosynthetically directed by the E. coli.
This sample can be called:
U-15N, 5% biosynthetically directed 13C (NC5) sample or
U-15N, fractional 5%-13C-labeled (NC5) sample
and give the Neri reference.
For RDC measurement:
- A secondary NC5 sample for RDC measurement.
(Alternatively, for the H-N RDC measurement, a [U-15N]-labeled sample can be used. Rutger's uses a second NC5 sample, since this is all ready being prepared for the stereospecific assignments, and a 13C CHSQC can be compared with the [U-15N, 13C]-labeled sample, to check if the samples are the same.)
For each protein that is a dimer in solution an extra sample may be required in addition to the samples above:
- 1:1 mixture of natural abundance and [U-15N, 13C]-labeled samples, for intermolecular NOE interpretation.
Typical Rutgers University NMR Buffers
The protein production facility at Rutgers University uses these three NMR buffers for the initial protein screening: :
- pH 4.5 NMR buffer: 20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
- pH 5.5 NMR buffer: 20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
- pH 6.5 NMR buffer: 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% NaN3, 5% D2O
Typical Rutgers University Protein Purification Protocol
E.coli BL21(DE3) are fermented in MJ9 medium . Cell pellets are stored at -20º C.
- add 30 ml Lysis buffer to the a frozen cell pellet and thaw.
- sonicate in ice bath
- centrifuge to remove insoluble part
- supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.
- exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore).
Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers
15N-labelled Glucose-based autoinduction media :
This is for 15N-labelling of samples for screening so we can take advantage of autoinduction during the screening phase when we are dealing with a large number of samples, but be able to switch to IPTG-induction with 13C-glucose as the sole carbon source for the smaller number of targets prepared for full NMR data collection, with high reproducibility.
For each sample, dissolve 6.8 g of Na2HPO4, 3g of KH2PO4, 0.5g of NaCl, and 0.6g of 15N-labelled NH4Cl, in 500 mL water and autoclave the media.
In a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO4 stock solution; 5 mg of biotin; 5 mg of thiamine.HCl; 10 uL of 1M ZnSO4 stock solution; 100 uL of 1M CaCl2 stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved 15N-labelled solution.
Follow the same fermentation protocol as with IPTG-induction method, except there is no need to monitor the cell density and omit the addition of IPTG. Grow the cells at 37C throughout.
Standard screening NMR buffers are:
|A5.0n300zd||Sodium acetate||300 mM||5.0|
|P6.5n450zd||Sodium phosphate||450 mM||6.5|
|P7.0n450zd||Sodium phosphate||450 mM||7.0|
All standard screening buffers contain 10 mM of the buffer, 10 uM ZnSO4, 10 mM DTT, 0.01% NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O.
The final NMR buffer for [U-15N, 13C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail.
(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).
Typical University of Toronto Protein Purification Protocol
i015t8.5n500z : 15 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4
i030t8.5n500z : 30 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4
i500t8.5n500z : 500 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4
- add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw.
- sonicate in ice bath
- spin down cell pellet
- transfer supernatant into new falcon tube and add 3 mL of nickel beads
- rock the tube for at least 30 minutes in cold.
- spin down the beads and discard the supernatant
- wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice
- in the final i030t8.5n500z wash, pour the beads unto gravity filter column
- elute the protein with i500t8.5n500z
- add benzamidine, and add DTT
- exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.
if it is [U-15N, 13C]-labeled, add step
(9a) put sample in dialysis bag with protease to cut his-tag and dialyse against cleavage buffer
(9b) pass the sample through nickel beads again, then follow step (10) above.
- ↑ Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” JACS, 127: 16505-16511. pmid = 16305237
- ↑ Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. J Biomol NMR 7: 131-141