Chemistry Reference
In-Depth Information
6.3 Membrane Protein Considerations
6.3.1 Quantity Limitations
Although TINS removes limitations such as size and solubility of the target protein to
be applied, there still remain quantity limitations with regard to membrane proteins. At
present, the practical lower limit for screening is roughly 25 μM solution equivalent (e.g.
nmol mL 1 settled bed volume). Since we typically prepare 500 μl of immobilized resin to
fill one cell of the sample holder, we require about 15 nmol of target. For a 50 kDa protein,
this works out to slightly under 1mg and therefore it is safe to use 1mg as a lower limit.
For soluble proteins in which structure-guided hit optimization is the primary means for
evolving fragments, this limit does not generally present a problem. However, for many
membrane proteins, formidable efforts are required to produce even this quantity. Accord-
ingly, efforts are under way in our laboratory to enhance the sensitivity of TINS towards
an eventual goal of being able to screen recombinantly expressed proteins in their native
membrane environment, that is, without purification. Below we present data demonstrat-
ing the feasibility of immobilizing such native membrane fragments. Since this approach is
beyond the present sensitivity limits of our TINS ligand screening station, however, current
efforts utilize highly expressed, purified and functionally solubilized membrane proteins.
Given the current requirement for about 1mg of functional protein to carry out ligand
screening, it is clear that an appropriate systemmust be available to produce large quantities.
Due to the interest in pharmacology and structure of membrane proteins, tremendous efforts
have been made in recent years in developing new means to express, purify and solubilize
them. It is not our intention to catalogue these approaches here, merely to mention some
which show promise with respect to producing sufficient quantities for ligand screening
and subsequent structural studies. Conceptually the simplest method for membrane protein
production is via cell-free expression. Recently, six different GPCRs have been produced
in milligram quantities using an E. coli -based expression system that included Brij78 as
a solubilizing detergent. [ 20 ] Studies were performed to show that at least one of the in
vitro expressed GPCRs was functional. Interestingly, all appeared to be dimeric. Bacterial
expression of membrane proteins typically results in the protein being unfolded and located
in inclusion bodies. Although purification of proteins from inclusion bodies is easy, the
requirement for refolding can represent a considerable hurdle. Nonetheless, companies
such as M-fold have successfully produced isotope-labeled GPCR using this approach and
showed that the protein was amenable to NMR studies. [ 21 ]
Beyond bacterial expression systems, a number of eukaryotic expression systems have
also been developed. One simple method of producing functional membrane proteins is
to generate recombinant transient or stable cell lines based on CHO or HeLa cells. Such
cell lines have the benefit of providing appropriate post-translational modifications such
as glycosylation which are not available in prokaryotic expression systems. [ 22 ] Often these
modifications are required for protein function, as shown for rhodopsin, where folding is
inefficient when the glycosylation site at its N-terminus is suppressed. [ 23 ] Unfortunately
the yield of proteins from stable cell lines is more often than not insufficient for ligand
screening studies. Transient expression of membrane proteins can increase the yield by
as much as a factor of 10, but results in other inconveniences such as repeatability issues.
Alternatives that have seen increasing success include recombinant expression in insect SF9
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