Escherichia coli, or
E. coli, has long been the trusty
workhorse of the structural biologist.
It is by far the most popular expression
host, that is an organism which is used to translate introduced DNA into
target proteins, with an astonishing 90% of structures deposited in the Protein
Data Bank (PDB) having at least one subunit if not the entire protein produced
by this bacterium. Targets are becoming
more ambitious, however, as the importance of larger protein complexes in
biological pathways becomes increasingly apparent. A recent review (Vincentelli, R., Romier, C. (2013)) examines whether E.coli
is fit to tackle these new challenges.
Can the workhorse be taught to jump fences?
E. coli as host for single expression
The virtual monopoly held by E. coli as a host is due to a number of factors, not least its ease
of use and low cost. It is amenable to
several different methods of genetic engineering, has rapid growth and benefits
from an ever expanding range of host specific tools.
There are mature methodologies available for the production
of a single protein. Reliable and
straightforward screening processes, which can discover the conditions required
for optimised protein solubility, are accessible for even challenging
targets. Since the solubility of a
protein has a direct impact on the size and quality of crystal that can be
grown, this is a critical aim.
Genomics laboratories tend to use robotic platforms which
can work with thousands of cultures side by side in order to optimise the
culture conditions for their long shopping list of proteins. This wealth of experience now allows
biologists to make a rational selection of a smaller set of expression
parameters when targeting proteins which are more difficult to produce. If this restricted method fails then the team
can revert to the broader set of conditions.
Supporting this trend towards more efficient screening
processes are studies that have identified the parameters with the greatest
impact on expression and solubility, a key example being specific fusion protein tags
which enhance solubility.
Another recent development which has increased the
complexity of proteins open to expression by E. coli, is the ability to co-express post-translational modifying
factors. These factors can be critical
in protein folding, complex assembly and catalysis, for example by promoting
glycosylations and disulphide bridges.
Streamlining for maximum
solubility and quality
The traditional approach to protein production was to
perform small scale expression tests designed to discover the conditions for
the highest soluble yield. These
conditions would be scaled up and only at this stage would consideration be
given to the quality of the sample, specifically protein aggregation,
oligomeric states, protein stability and correct folding.
By using growth media with high cell density, particular E.coli strains and protein fusions
designed to enhance solubility, initial yields have been increased. This coupled with the improvement in
biophysical characterization, such that assays to examine protein quality can
now be performed on just micrograms of a sample, has meant that culture conditions
can be optimised for both solubility and quality at the first stage thereby
streamlining the expression protocols.
E. coli and the expression of macromolecular complexes
Most biological processes involve macromolecular complexes
alongside single proteins and there is an increasing desire to understand the
structural and biochemical basis of these larger structures. The co-expression of partner proteins has
been demonstrated to be advantageous for complex formation as protein-protein
interactions are often the platform which allows co-folding and
co-stabilization.
One example is the fimbrial tip complex of E. coli.
Many Gram-negative and some Gram-positive bacteria are covered in a
fringe of short, thin fimbriae which are used to attach both to eukaryotic
cells and to each other although the mechanism was previously unknown. Co-expression of the FIM proteins of the
fimbrial tip established that each subunit inserts a β strand into its
neighbouring subunit such that allosteric changes in the tip protein trigger
signals which can be passed down the fimbria.
 |
Image adapted from (Le Trong, I. et al., (2010)). A view of a fimbrial tip complex of E.
coli. (PDB 3JWN)
|
Birkbeck’s head of biological sciences, Prof Gabriel Waksman, has
also used this technique to elucidate the interaction of FimH with its
transmembrane translocation channel. His
work has been the subject of previous blogs in June
’11 and May
’08.
Another impressive example which illustrates the size of
complex that can be achieved through co-expression is the 1.8MDa baseplate of
the lactococcal phage TP901-1. The
baseplate is responsible for adhesion of the phage to the host and for delivery
of the genome at infection and this particular version consists of 6 subunits
of DIT, 18 of BppU and 54 of RBP proteins.
 | |||||
Image adapted from (Veesler, D. et al. (2012)). The baseplate of the lactococcal
phage TP901-1. (PDB 4DIW)
|
Despite the advantages
of co-expression, only a small percentage of large complexes listed in the PDB
have been fully produced this way.
Often, subunits are produced using co-expression but then labour
intensive in vitro reconstitution
strategies are employed to form the complete complex.
In some cases, this is because the protein complexes have
critical interactions with nucleic acids but this is not always the case. This
raises the question of what the barriers are that are discouraging complex
formation via co-expression from E.coli.
Approaching the jumps
Studies have found various parameters that influence the
quality and yield of co-expression using E.coli. Results can vary depending on whether a
single vector is used to introduce the target genes rather than multiple
vectors, whether multiple genes are used rather than cis and trans copies of
the same gene or on the precise location of the affinity tag.
The best approach to tackle this quantity of possible sets
of conditions is to use the high-throughput technologies which have been refined
so effectively for single expression protein production. This will need to be combined with the tools
in development for miniaturization of biophysical characterization so that the
sample quality can be considered during the initial stages rather than creating
a further bottleneck as a second round of tests are performed with greater
quantities.
As techniques for performing characterization assays on
minute samples improve further and, hopefully, a co-expression system can be
evolved which allows the production of protein/RNA and protein/DNA complexes,
there is optimism that E. coli can
extend its hosting duties into ever larger and more intricate protein
complexes.
Protein-protein interactions and protein expression for
structural biology is covered in detail in the TSMB course.
