Biomedical Engineering Reference
In-Depth Information
in Section 12.7, such studies can provide extremely useful information on, for
example, ligand binding without the need for full structure determinationas
well as dynamics of protein regions. Furthermore, the potential to carry out
such studies in a more native environment should be of great value.
This chapter aims to assess the current state-of-the-art of solution NMR in
this field, whilst providing a critical assessment of the challenges and potential
of such techniques. Overall, it seems clear that solution NMR has the proven
ability to provide extremely relevant information both in the field of full
structure determination of membrane proteins and through functional studies.
12.2 Protein Expression
A major challenge in any biophysical study of membrane proteins is the
production of sufficient, functional protein. For full structural investigations,
this is a particular problem where mg quantities of protein are required.
Difficulties arise due to the need to target the protein to an appropriate cellular
membrane compartment where it can fold correctly, or where it can be
protected from proteolytic degradation before refolding is attempted in vitro.
Even if protein can be expressed, the functionality of the protein must be
verified since this can vary in different expression hosts. These difficulties
increase for expression of the key class of G-protein-coupled receptors; away
from their native mammalian hosts, these receptors are frequently expressed at
very low yield, degrade rapidly and may not be functional. A variety of
expression systems are available. To date there is no clear leader, and
expression is typically approached on a case-by-case basis, making this one of
the most time-consuming aspects of membrane protein studies. Nevertheless,
considerable work has been done in this field, providing a number of new and
potentially extremely promising methods to overcome expression hurdles,
from which recent crystal structures including of GPCRs have benefitted, and
it is expected that this hurdle will become increasingly easy to cross. However,
in comparison, NMR studies have the added requirement of isotopically
labelled protein, which may constrain the choice of expression system.
12.2.1 Escherichia coli
This system has been well developed for soluble proteins, providing a wide
variety of vectors and tags as well as purification and refolding techniques. For
NMR studies, it also has the considerable advantage that labelling studies are
well developed, enabling
15
N,
13
C, and
2
H labelling as well as selective amino
acid and non-natural amino acid labelling, which can be achieved through
incorporation of labelled precursors. Strains mutated in certain biosynthesis
genes can improve the selectivity of the uptake whilst auxotrophic strains may
also be used to incorporate specific amino acids.
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Furthermore, these
approaches are relatively cost-effective and E. coli expression can be easily
scaled up once appropriate conditions are found.
