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184 Pattenden and Thomas
binding efficiency is reduced using 10% glycerol but appears to be tolerated, the
binding efficiency is significantly reduced below 5% in the presence of 0.25%
Triton X-100 or Tween 20, and is completely precluded in the presence of 0.1%
SDS. It is advised not to use a detergent as an additive to assist lysis as varied
results occur, however if used, the binding efficiency is often suitably restored by
dilution prior to binding to the amylose matrix (2) (∼0.05% Tween 20 and ∼1:10
dilution for B-Per retains ∼80% binding efficiency). The authors have found
there can be batch-to-batch inconsistencies using B-Per extraction reagent that
could be related to detergent effects dependent on MBP-passenger protein and
total protein concentrations. We have specifically noted proteolysis inefficiencies
following detergent extractions.
6. Under normal conditions defined as 15 mL of amylose agarose matrix processing,
1 L of Lauria Bertani media supplemented with 0.2% glucose (producing ∼40
mg MBP fusion protein); the deterioration of the matrix is reported to be approximately
1–3% of the initial binding capacity each time it is used. It is stated
that such a column may be used up to 5 times before a decrease in yield is
detectable (5–15% lost binding capacity), and up to 10 times before the loss is
significantly noticeable (10–30% lost binding capacity). However with different
media producing heavier cell densities but a lower MBP fusion protein yield, the
loss of amylose binding capacity will be more dramatic.
7. The inhibitor cocktail is a solution containing protease inhibitors to reduce
the degradation of the recombinant protein due to the activity of proteases
released from the bacterial cell upon lysis. They generally consist of
broad specificity inhibitors of serine, cysteine, aspartic and aminopeptidases,
with the activity of EDTA and EGTA influencing metalloenzymes
and proteases (see Note 8). Inhibitor cocktails can be purchased from most
chemical supply companies or made in-house using a combination of nonspecific
and/or specific protease inhibitors. The Expasy peptide cutter tool
(http://au.expasy.org/tools/peptidecutter/) (35) can be used to predict potential
proteolysis issues or specific protease classes which may be an issue to a given
MBP-passenger protein amino acid sequence. Using the peptide cutter tool, a
particular set of potential proteolysis issues can be identified and addressed using
protease inhibitors. In general, inhibitor cocktail comprise phenylmethylsulfonyl
fluoride (1 mM), aprotinin (1 μg/mL), leupeptin (1 μg/mL) and pepstatin A
(1 μg/mL) in the buffer. Specific care should be taken with washing steps
following protease solutions if proteolysis is to follow purification.
8. EDTA and EGTA chelate metal ions that are important to metalloproteases and
metalloenzymes. EDTA specifically chelates divalent and trivalent metal ions
such as Mn(II), Cu(II), Fe(III) and Co(III). EGTA has a higher affinity for Ca(II)
compared to EDTA, and calcium ions may be particularly relevant to MBP
purification as a co-factor for some formulations of Factor Xa used to separate
MBP from the passenger protein, but also as a known cofactor for potential
contaminants of the maltose regulon (18).