Biology Reference
In-Depth Information
eter and produce between 0.2 and 1 L of medium depending upon expression
levels and experimental requirements.
1.
Medium is harvested and stored at -20
°
C until required.
2.
Upon thawing, the protease inhibitors NEM and PMSF are added and as both are
sensitive to hydrolysis they are readded fresh every 24 h the protein is maintained
above freezing. To further reduce degradation, EDTA is added to give a final
concentration of 10 m
M
. All subsequent purification steps are carried out at 4
°
C.
3.
Where large volumes are being used, the protein solution may be concentrated
5 to 10 times using an ultrafiltration cell (Amicon, Danvers, MA) with a mem-
brane pore size chosen according to the molecular mass of the recombinant pro-
tein (protein binding to the membrane may be reduced by initially rinsing the
membrane in 5% Tween-20 overnight and then rinsing it extensively with water
before use).
4.
The StrepTactin column is equilibrated with five column volumes of washing
buffer passed through at 0.2 mL per min for a 1-mL column.
5.
The protein is loaded on the column at the same flow rate and is recirculated over
12-24 h.
6.
The column is washed with at least five column volumes of washing buffer at
0.2 mL per min.
7.
Elution of the protein occurs upon the addition of three column volumes of elu-
tion buffer and 0.5-mL aliquots are collected and analyzed (
Fig. 2A,B
).
8.
The column is cleaned using 15 column volumes of HABA-containing regenera-
tion buffer. HABA has a yellow color and displaces the desthiobiotin. Upon
interaction with avidin homologs, it has a color shift from yellow to red that can
be used to indicate the removal of desthiobiotin from the column.
9.
Finally, the column is washed free of the HABA using 10 column volumes of
the wash buffer. The matrix should now loose its red pigmentation. The column
can be stored at 4
°
C when maintained in wash buffer containing 0.02% w/v
sodium azide.
10.
A number of problems with recombinant protein purification may be encoun-
tered, and these are discussed in
Notes 3-5.
4. Notes
1.
Tags may not always be innocuous
(8)
.
We have produced the CMVNstrep with
protease sites for factor X, enterokinase, or thrombin to allow the removal of the
Strep tag if it is felt to be a problem for later studies.
2.
New batches of puromycin should be tested for their activity, to determine the
minimum concentration able to give total cell death after 5 d of selection of the
untransfected control.
3.
Low or no expression of the protein may be caused by a number of causes.
a. Frame shifts either owing to an error in choosing primers or an artifact in cloning.
b.
A PCR-induced mutation introducing a stop codon or leading to a major amino
acid substitution affecting the structure of the expressed protein and leading
to its intracellular degradation.
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