View - ResearchGate

172 Pattenden and Thomas
functions as a molecular bivalve; the protein adopts two conformations: an
unliganded ‘open’ conformation and a ligand-bound ‘closed’ conformation that
involves a twisting rotation of approximately 8° and bending movement of up
to 35° by the N-terminal lobe (26). The residues forming the binding cleft are
placed at the surface of the two domains, so upon binding, the ligand induces
the conformational changes that allow the two globular domains to enclose
the ligand-binding cleft, excluding solvent and forming a stable, bound state.
Positioned behind the ligand-binding cleft is a hinge region, which facilitates
the opening and closing structural movements that occur with ligand-induced
conformational change.
Fusion constructs of MBP are not normally engineered with the passenger
protein at the N terminus, as such constructs are not frequently soluble and
do not readily purify. The N-terminal region is located external to the ligandbinding
cleft but undergoes radical changes upon ligand binding. Therefore,
steric or thermodynamic effects may occur with N-terminal constructs to
influence the conformational changes in this region of MBP depending on the
size and nature of the construct, impinging on the ability of MBP to open
and close – precluding binding to the amylose affinity resin (27). Therefore
the C terminus is the preferred site of cloning and appears to undergo far less
structural changes in response to ligand binding (26).
1.1. Aspects of MBP and Amylose Biology and Chemistry
that Impact on Purification
For E. coli expression of MBP, the history (including codon usage) and high
intrinsic concentration of MBP are features of the biology, making MBP very
favourable as a carrier protein for heterologous expression – depending on the
nature of the cloning and physico-chemical properties of the passenger moiety.
There are two different construct types available from New England BioLabs.
The first type allows for typical recombinant E. coli expression localized in
the cytoplasm at large levels. The second construct-type exploits MBP biology
to direct expressed proteins to the periplasmic space at modest levels, but
provides a simplified bioprocess (see Note 3) from a unique environment where
native disulphide bonds may be formed and a proteolytic profile exists which
is distinct from the cytoplasm.
New England BioLabs claim purification ranges, as high as 200 mg/L culture
have been obtained for MBP fusion proteins with typical yields in the range
of 10–40 mg/L culture for cytoplasmic expression (being 20–40% of the total
cellular protein), while typical yields from periplasmic expression being ≤4
mg/L culture (1–5% of the total cellular protein) (2). It is not uncommon to
obtain yields of 100 mg/L from shake-flask cultures (28). With favourable