Protein purification

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.

1. add 30 ml Lysis buffer to the a frozen cell pellet and thaw.
2. sonicate in ice bath
3. centrifuge to remove insoluble part
4. supernatant is added to an AkTAxpress^TM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.
5. 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:

name	buffer	NaCl concentration	pH
A5.0n300zd	Sodium acetate	300 mM	5.0
Bt6.0n450zd	bis-tris	450 mM	6.0
Bt6.5n450zd	bis-tris	450 mM	6.5
M6.5n450zd	MOPS	450 mM	6.5
P6.5n450zd	Sodium phosphate	450 mM	6.5
P7.0n450zd	Sodium phosphate	450 mM	7.0
H7.0n450zd	HEPES	450 mM	7.0
H7.5n450zd	HEPES	450 mM	7.5
T7.7n500zd	tris	500 mM	7.7

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

Required buffers:

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

1M DTT

1M benzamidine

1. add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw.
   
2. sonicate in ice bath
3. spin down cell pellet
4. transfer supernatant into new falcon tube and add 3 mL of nickel beads
5. rock the tube for at least 30 minutes in cold.
6. spin down the beads and discard the supernatant
7. wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice
   
8. in the final i030t8.5n500z wash,  pour the beads unto gravity filter column
   
9. elute the protein with i500t8.5n500z
10. add benzamidine, and add DTT
    
11. 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.