A I40 Biochemical Society Transactions ( 1999) Volume 27, part 5
D2 "Expression and purification of the
mitochondrial
Uncoupling Proteins (UCPs): a comparative study between
Escherichia coli and Sacchuromyces cerevisiue."
Bruno Miroux 8, t
5 CNRS-CEREMOD, 9 r. J . Herzel. 92190 Meudon. France
e-mail: [email protected]
t Laboratory of Molecular Biology. Hills road.CB2 2QH Cambridge, UK
Uncoupling proteins belong to the mitochondrial anions carrier
family and are located into the inner membrane of mitochondria.
Using a library of fusion proteins expressed in E. coli,
monospecific antibodies were selected from a whole serum antiUCP, and were used to establish the orientation of the
corresponding region of the protein inside or outside the
mitochondria. Our results showed that UCP contains six
membrane spans, both extremities facing the cytosol. The full
length UCPl was as well functionally expressed in
Saccharomyces cerevisiue. Mitochondrial uncoupling activity was
assayed by oxygen consumption, membrane potential
measurement and by flow cytometry (Arechaga et al. 1993). The
yeast expression system proved to be very efficient for functional
analysis and mutagenesis of the UCPs but, so far, the amount of
protein produced is low. Other mitochondrial carrier have been
expressed in E. coli but the amount of protein produced was
limited by the toxicity of the expression. Using the T7 RNA
polymerase based expression system, a method to optimise overexpression of proteins in E. coli was developed. Bacterial
mutants were selected for their ability to overcome the toxicity
associated with the over-production of the oxoglutarate carrier.
Analysis of the mutant hosts showed that modulation of the
transcription is critical for the optimisation of the over-production
of the target protein (Miroux and Walker 1996). All UCPs are
now highly expressed in the new mutant strains opening the way
to large scale refolding of the inclusion bodies. Both yeast and
bacterial expression systems are in competition for the production
of the large amount of protein required for structural studies.
Advantages and disadvantages of each one will be discussed.
D3
D4 FUNCTIONAL EXPRESSION IN E. COLI AND
PURIFICATION OF A G PROTEIN-COUPLED
RECEPTOR FOR NEUROTENSIN
Reinhard Grisshamme r, MRC Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 2QH, U.K.
G protein-coupled receptors (GPCRs) are integral membrane
proteins and important pharmacological targets. Our goal is to
understand their molecular structure and action. We have
expressed a receptor for neurotensin (NTR) in Escherichiu coli
because this receptor does not occur naturally in large quantities
(see Grisshammer & Tate, Q. Rev. Biophys. 28, 315-422,
1995). We successfully increased NTR expression to 1000
copies per cell in functional, membrane-inserted form by using
a fusion approach, where maltose-binding protein with its
signal peptide is linked to the receptor N-terminus and a decahistidine tag to its C-terminus, and by refining growth
conditions. NTR was purified to homogeneity by imrnobilised
metal affinity chromatography followed by a neurotensin
column (Tucker & Grisshammer, Bioche-n. J. 3 17, 89 1-899,
1996; Grisshammer & Tucker, Protein Expression and
Purification 11, 53-60, 1997). We obtain now 1-2 milligrams
of functional receptor protein from 10 L of culture. This
purification scheme sets the stage for structural studies of NTR
and we are currently in the process of exploring conditions for
formation of two-dimensional crystals. This work is supported
by the Medical Research Council, GlaxoWellcome and Zeneca
Pharmaceuticals.
Expressionof prokaryotic membrane transport proteins in
Escherids d i successasand failures.
-and
Peter J.F. Henderson
Schod of BEhemistty and Molecular Biobgy, University of Leads.
Leeds. LS2 9JT
The arrplified expression d membrane proteins has often proved to be
-
d f i l t . However, we have wccessfully expressed prokaryotic
membrane transport proteins. using Escherichia coli (E. mli) expression
system, to 20-50% d the inner membrane protein. Using the plasmid
plTOl8. genes for prokaryotic sugar, amim acid and mulidrug
resistance membrane transport proteins have been cbned under the
Contrd d a tac prwnoter that is repressed in the absence of IPTG by the
plasmid encoded Lac repcessoc. The E. coli galactose-H' symport protein
(Gal@ and the prolinehetaine transporter (Prop) have been
overexpressed in plasmid pBR322 using the galP promoter for
constitutive expression. The arabinose-H' (AraE) and fucose-H' (FucP)
symporters have been cloned under the control of the tightly regulated 1
P, promoter in plasmid pAD2847 and overexpression induced by nalidiiic
acid to remove repression by c I produced by the host strain AR120.
Inevitably the expression d some membrane proteins in E. coli is
associated with toxicity and ceU death eg: the norfloxacin resistance
protein. NorA. from Staphy/ocmus aureus. In such cases choice of
media, dl host. titration of inducer and time of induction can al aid
overexpression. After membrane preparation. overexpressed proteins
am detected by SDS-PAGE gel electrophoresis, Westem blot and K
terminal sequencing. To date al proteins analysed migrate with an
anomalously low apparent Mr. Hexahistidine tags have been routinely
added at the C-terminus, which is believed to minimise incorrect folding
and optimise insertion of the protein into the membrane, for affinity
purification using NcNTA chromatography. Analyses of purified and
reconstituted material by circular dichroism and fourier transform infm red
spectroscopy indicate that the proteins contain 40-90% a-helix. Activity
measurements, camed out in reconstituted systems. of both wild type
and mutant proteins have albwed insights into key residues that are
important for the activity of the protein. There has only been one protein
refractory to overexpression; the E. mli ~ - h m n o s e transporter
(RhaT).This may be due to the N-terminus having a periplasmic. rather
than cytoplasmic, location.
0 I999 Biochemical Society
D5
Overexpression, Refolding, and Crystallisation of an 80 kD Outer
Membrane Iron Transporter from the Outer Membrane of E. colr
S.K.Buchanan
Departmenr of Crystallography, Birkbeck College, Malet Street, London
WCIE ?HX, UK
Ferric enterobactin receptor (FepA) IS an 80 kD outer membrane protein
from E. coli which binds ferric enterobactin (719 daltons) and transports it
into the periplasm; it also serves as the receptor for colicins B and D. FepA
belongs to the family of high affinity, active transport receptors which
specifically transport iron chelates and vitamin B,, across the outer
membrane. All members of this famliy derive energy for transport from the
proton motive force across the inner membrane, and this is accomplished
b y periplasmic contact with an integral inner membrane protein complex,
TonB-ExbB-ExbD.
FepA has been overexpressed in the cytoplasm of E. colr, producing large
quantities of insoluble inclusion bodies. The inclusion bodies were solubilised in urea and refolded using a combination of sulfobetaine 3-14 and
sodium dodecylsulfate. The refolded protein was subsequently purified by
FPLC using anion exchange and gel filtration chromatography. Refolded
FepA was crystallised according to methods developed for native
(membrane-inserted) FepA; the resulting crystals have the same space group
and unit cell dimensions determined for native FepA crystals. The current
yield of refolded protein is approximately 10 mg/l cell culture, making this
method suitable for structural studies of other outer membrane proteins.