Shweta Singh
2
Adrien Gras
2
Cdric Fiez-Vandal
1
3
Jonathan Ruprecht
2
Rohini Rana
2
Magdalena Martinez
2
Philip G Strange
0
Renaud Wagner
[email protected]
1
Bernadette Byrne
2
0
School of Pharmacy
,
PO Box 228
,
University of Reading
,
Whiteknights, Reading RG6 6AJ
,
UK
1
Unite Mixte de Recherche 7175 Ecole Superieure de Biotechnologie de Strasbourg - Centre National de la Recherche Scientifique, Departement Recepteurs et Proteines Membranaires
,
67412 Illkirch
,
France
2
Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London
,
South Kensington, London, SW7 2AZ
,
UK
3
Karolinska Institutet, Department of Medical Biochemistry and Biophysics (MBB), Division of Biophysics
,
Scheeles vag 2, 171 77 Stockholm
,
Sweden
Background: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Results: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a Cterminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A2AR, and therefore more suitable for further functional and structural studies. Conclusion: Large-scale expression of the A2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.
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Background
G-protein-coupled receptors (GPCRs) form a large
superfamily of cell-surface receptors that mediate cellular
responses to a wide range of biologically active molecules
including hormones, neurotransmitters and drugs.
Indeed, half of all currently available pharmaceuticals act
through GPCRs [1,2]. The physiological and
pharmacological importance of these proteins makes them key
targets for drug discovery programmes. Our understanding
of the precise mechanism of action of these important
proteins is currently limited by a lack of high-resolution
structural data. One limiting factor to structural studies of
GPCRs has, until recently, been low expression levels [3].
With the exception of rhodopsin, all GPCRs are expressed
at very low levels endogenously, thus requiring the
development of recombinant overexpression systems. Careful
expression vector design, GPCR codon-optimisation [4]
and high throughput approaches used to identify GPCRs
with the highest expression levels in different expression
systems [5] are among the methods that have been used
to produce sufficiently high levels of functional GPCRs
suitable for structural studies.
Success has been achieved when using the expression host
Pichia pastoris for the production of membrane proteins
for structural studies, most notably the rat membrane
protein K+ channel [6]. Pichia has several advantages over
other systems for the production of GPCRs. It is easy to
manipulate, has high production levels and is relatively
inexpensive. In addition, Pichia has the ability to
glycosylate expressed receptors, albeit in a modified form
compared to higher eukaryotes, which is essential for the
proper functioning and membrane targeting of many
receptors [7-10]. Much effort has been applied to the
optimisation of Pichia expression systems specifically for
GPCR production [11-16]. The basic system uses a
pPIC9K vector (Invitrogen) where GPCR expression is
under the control of the strong methanol inducible AOX1
promoter. Protease deficient expression strains, such as
the SMD1163 strain, and the use of the -factor leader
sequence have improved receptor expression levels
[11,12]. Modifications to the growth media including
addition of histidine, receptor specific ligands and
di (...truncated)