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182 Pattenden and Thomas
8. Collect 1 mL fractions and elute the MBP-passenger protein with 15 CV of elution
buffer.
9. Regenerate the column using the steps described in Subheading 3.5.
3.5. Regeneration Conditions for Amylose Agarose or Amylose High
Flow Matrices
1. Wash the matrix with 5 RV/CV of final wash or column buffer (see Note 19).
2. Wash the matrix sequentially with 5 RV/CV of regeneration 1 and regeneration 2.
3. Wash the matrix with 10 RV/CV of ddH 2 O (regeneration 3).
4. Wash the matrix with 5 RV/CV of regeneration 4 and store at 4°C.
3.6. Matrix-Assisted Dialysis Refolding Methods
1. Lyse bacteria in no more than 5 mL of denaturation buffer (see Note 11) and form
a cleared lysate.
2. Place cleared lysate in a suitable dialysis cassette or membrane (10–30 kDa cutoff).
3. Dialyze at 4°C in 1 L (refold 1) for 8 h.
4. Transfer to 1 L (refold 2) and dialyze for 8–16 h.
5. Transfer to 1 L (refold 3) and dialyze for 8 h.
6. Remove from dialysis membrane (see Note 20) to a suitable vessel and proceed
as for batch/column method (Subheading 3.3., steps 7–23).
4. Notes
1. MBP (New England BioLabs pMAL-C2 construct calculated as a Factor Xa
cleaved product) has a molecular weight of 42,481.9 Da, an acidic theoretical pI
of 5.08, a molar extinction coefficient of 1.541 M/cm (A 280 nm 0.1% = 1 g/L)
and favourable aliphatic index of 80.78 (35). The authors have found the molar
extinction coefficient is not an accurate means to estimate MBP fusion protein
concentration in non-denatured solutions and could be related to a change in
spectral fluorescence noted at longer wavelengths (a tryptophan red shift) with
conformational changes upon ligand binding (36). The aliphatic index indicates
the relative volume occupied by aliphatic side chains is quite high in the protein
and is a positive factor for increased thermostability (35,37), which may allow
a protein to more easily refold by allowing the protein to undergo a rapid and
stable hydrophobic collapse to conformations close to the native state (38). The
thermostability and refolding ability of MBP has been noted in the literature
and is maximal at pH 6 (T m of 64.9°C, H m of 259.7 kcal/mol) (19,20,39).
MBP is stable between pH 4 and 10.5 (25°C) and undergoes a reversible, twostate
refolding mechanism at neutral pH in the presence of temperature variation
and chemical denaturants (22,39). The association constant (K a ) for a range of
maltodextrins to MBP is between approximately 2 and 4 × 10 −7 M/s, and so