Protein purification

Samples for NMR

For each protein, we usually make the following samples:

•    100% 15N, 100% 13C uniform labeled sample, for resonance assignment and NOE interpretion.
•    100% 15N, 5% 13C labeled sample, for stereospecific assignment of VAL and LEU isopropyl moieties.

For RDC measurement:

•    A secondary 100% 15N, 5% 13C labeled sample, for RDC measurement.

For each protein that exist as dimer in solution an extra sample may be required in addition to the samples above:

•    1:1 unlabeled and 100% 15N, 100% 13C uniformed labeled mixed sample, for intermolecular NOE interpretation

Typical Rutgers University NMR Buffers

The protein production facility at Rutgers University uses three typical NMR buffers for the initial protein screening.

They are :

• 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 -20 degrees.

(1) add ? to the a frozen cell pellet and thaw.

(2) sonicate in ice bath

(3) centrifigure to remove insoluble part

(4) supernatant is added to an AkTAxpressTM 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) Buffers

standard screening NMR buffers are:
a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

final nmr buffer for 13c/15n labelled 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

before you start, buffers needed:

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 13c/15n sample, 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.