Transcript Document

Expression and Purification of Integral Membrane Proteins from Yeast for the
Center for High-Throughput Structural Biology
*
Clark ,
*
Fedoriw ,
*
Robinson ,
†
Sullivan ,
Kathy
Nadia
Katrina
Mark
Michael G. Malkowski‡, George T. DeTitta‡, and Mark E. Dumont†*
*Department of Pediatrics and †Department of Biochemistry and Biophysics University of Rochester Medical Center
Rochester, NY 14642 and ‡The Hauptman-Woodward Institute, 700 Ellicott Street, Buffalo, New York 14203
Target selection
Vectors for yeast membrane protein expression
Summary
To address the severe lack of three dimensional structural information for eukaryotic
transmembrane proteins (TMPs), the Center for High-Throughput Structural Biology is developing
protocols for expression and purification of TMPs in the yeast Saccharomyces cerevisiae. We have
focused initially on a set of endogenous yeast TMPs that are the highest expressing reading frames
in a previously-constructed genomic collection of S. cerevisiae expression clones and for which there
are established biochemical assays for determining whether the protein is maintained in a native
state. Genes encoding the target TMPs are transferred via ligation-independent cloning procedures
to a series of vectors that allow galactose-controlled expression of reading frames fused to Cterminal His6, His10, and ZZ (IgG-binding) domains that are separated from the reading frame by a
cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have
been purified via affinity chromatography and gel filtration chromatography at levels and purities
sufficient for ongoing crystallization trials. Single chain antibodies (scFvs) recognizing several
targets have been developed as aids to crystallization and purification. Current efforts are focused on
overcoming bottlenecks in protein production and crystallization by introducing the following
improvements at different levels of the production pipeline: 1) improving overall levels of cellular
expression of TMPs by altering protocols for cell growth and induction of expression; 2) increasing
efficiency of cell lysis; 3) increasing the efficiency of detergent solubilization; 4) increasing the yield of
3C protease cleavage; 5) reducing the number of steps required for effective purification; 6)
optimizing the amount of residual lipid purifying with the TMP; 7) developing protocols that allow
production of highly concentrated protein solutions that do not also contain high detergent
concentrations; 8) the use of additives such as lipids and enzyme inhibitors to stabilize purified
proteins.
Targeting Strategies
30 Target ORFs are currently selected based on the following criteria:
MORF library vector (Gateway cloning)1
PGAL
ATT site
ORF
ATT site
His6
HA
3C
1. Prediction of two or more transmembrane segments based on TMHMM and HMMTop
ZZ
ORF cloning
pSGP36 (Ligation independent cloning)
PGAL
PGK1 5’ LIC site
ORF
2. Absence of evidence that ORF is part of a hetero-multimeric complex, based on
genomic/proteomic databases.
3. High level expression in C-terminal-tagged genomic Saccharomyces cerevisiae MORF
library of Gelperin et al. (2005). (263 predicted integral membrane proteins in MORF
library are expressed at levels of ~1mg/l. Of these, 90 have human orthologs)
LIC site 3C His10
pSGP40 (Ligation independent cloning)
PGAL
PGK1 5’ LIC site
ORF
LIC site 3C
4. Existence of a published procedure for assaying native state of produced protein.
ZZ His10
Fermentor culture
(autoinduction galactose)
Culture conditions: Issues
KCl-stripping of membranes
S. cerevisiae achieves >100 g/liter (dry cell weight) in fermentation on rich media
BUT: Plasmid losses of ~50% are observed for some of our strains on rich medium
ALSO: We find that growth at low temperatures (26oC) stabilizes some membrane
proteins against subsequent precipitation.
Un-stripped membranes
0.7 M KCl-stripped membranes
Harvest, lyse (Avestin)
YNL275w-40
3,000 x g spin
Talon-binding proteases of yeast
(solubilized protein) Pellet
1-Step Purification of
Ste24p (CAAX
protease) on Talon
49 kDa
3C-cleavage site
5. Also: Use of Nickel-NTA resin inhibits
subsequent 3C protease cleavage whereas use of cobalt (Talon) does not.
3C protease
cleavage
Imidazole
elution
Static Light Scattering
Crystallization trials
100 kDa filtrate
Concentrate
100 kDa concentrate
Gel filtration
50 kDa filtrate
0.1 M NH4Br 0.1M Acetate pH 5
20% PEG 8000
50 kDa concentrate
0.1 M NH4Br 0.1M Tris pH 8
20% PEG 8000
5 ul
Ste24p expressed from vector
pSGP40 was solubilized from KClwashed membranes, bound to Talon,
then eluted by cleavage with His6tagged 3C protease. After elution, the
Talon column was treated ith 500 mM
imidazole to visualize
Purification from
96,000 OD mls
Concentration of purified
protein in the presence of
detergent
Anion transporter YNL275w (pSGP40, cleaved)
0.2 M KSCN pH 7
20% PEG 3350
3C-GST protease
( 5 ug)
Detergent exchange
and dilution
tag (Z-domain)
3C-6HIS protease
( 7 ug)
His-His-His-His-His-His
10 ul
Bind to IMAC or IgG
affinity matrices
20 ul
3. Inefficient cleavage can sometimes be
overcome by adding large amounts of protease.
500 mM imidazole
elution
detergent
3C-His6 elution
protein
Marker
3C-GST 5 g
Ynl275w 5 l
Yln275w 10 l
2:26:46.4
Marker
1:38:14.4
Sup
Salt-washed membranes
3C-His6 elution
Markers
1:23:12.0
1:14:08.0
Elutions after GST resin
IgG stripped urea/SDS
IgG Elution 3
IgG Elution 2
IgG Elution 1
IgG super rebound to IgG
Urea/SDS stripped Talon
Talon Elution 3
Talon Elution 2
Talon Elution 1
1. High-purity yeast transmembrane proteins are now being produced for crystallization and have
successfully served as antigens for generating recombinant single chain antibodies for cocrystallization. The best yields of purified protein are 0.3 mg/l of culture.
2. The goal of “E. coli-fying” yeast as an expression system for membrane proteins will benefit from
ongoing development of improvements in the following areas:
- Development of culture and induction conditions leading to increased overall expression of folded
proteins.
- Use of repeated cycles of cell lysis for more complete recovery of targets.
- Selection of optimum detergent for efficient solubilization based on recent genome-scale surveys
of detergent effectiveness such as that of White et al. (2007).
- Development of purification protocols that do not rely on cleavage of tags or engineering of specific
proteases with enhanced activity toward detergent-solubilized proteins.
- Development of rapid purification protocols that maintain a population of protein-bound lipids.
- Maintenance of high protein concentration throughout purification to avoid extensive concentration
of detergent in final steps.
EDTA-Stripped
Current bottlenecks/solutions
1.2M KCl;
120,000 x g spin
Detergent solubilization
26,000 x g spin
2. The activity of 3C protease is not
intrinsically sensitive to detergents.
4. Affinity tags on yeast membrane proteins
do not appear to be as accessible as the
same tags on soluble proteins (His10 is
useful but His6 generally is not.)
500 mM imidazole
02:00:00
Hr:Min:Sec
300 mM imidazole
01:00:00
150 mM imidazole
00:00:00
50 mM imidazole
psi
15 mM imidazole
-100.0
5 mM imidazole
-0.010
275W-40, from IgG
0.0
Marker
0.000 0.0
Ste24p stripped from Talon
using EDTA
EDTA-Stripped
0.010
100.0
Ste24p cleaved from Talon
with GST-tagged 3C protease
Membrane Pellet
500 mM imidazole
0.020
Endogenous yeast proteases
that degrade the Ste24p target as
well as 3C protease include
protease B (Prb1p) and can be
inhibited by PMSF (but not all
serine protease inhibitors.)
Sup
300 mM imidazole
200.0
0.030
3C-GST
150 mM imidazole
300.0
0.050 50.0
0.040
Strain 1: BJ5460 pep4- prb1
Strain 2: EJG1117 pep4- prb1Strain 3: EJG1364 pep4- PRB1+
50 mM imidazole
0.060
Ste24-40
cleaved
15 mM imidazole
0.070
100,000 x g spin
Ste24-40
uncleaved
1. Many tagged yeast membrane proteins
are not efficiently cleaved by 3C protease
400.0
Ynl275w
-
Pellet
5 mM imidazole
2 6 10 16 22 28 34 40 46 52 58 64 70 76 82 88 94 100 107 114 121 128 135 142 149 156 163 170
0.100
500.0
100.0
% Buffer B
0.090
Gel Filtration
Superdex 200
Lysate
Each purification:
300 OD mls
The C-terminal tags of many yeast membrane
proteins may be obscured by detergents
Fraction
#
Fractions
0.080
+ - + - +
3
Wash 2
3C-GST
Detergent: dodecyl maltoside
Culture: 96,000 ODmls
2
Wash 1
Ynl275w
(cleaved)
1
Marker, 15 uL
Multi-step purification of the
anion transporter YNL275w
1
2
3
Strain
PMSF + - + - + -
275W-40 from IgG
1. To date, only three structures of heterologously expressed eukaryotic transmembrane proteins
have been solved by x-ray crystallography. Both of these proteins were expressed in yeast.
2. Advantages of homologous expression system for post-translational modifications, membrane
targeting, protein folding, lipid requirements
3. Extensive annotation of yeast genome as far as protein-protein interactions, subcellular
localization, expression levels, protein function
4. Availability of yeast strains with altered protein degradation, unfolded protein response, posttranslational modifications, intracellular trafficking
5. Rapid and inexpensive conditions for culturing yeast cells
Loading: 1/200th
ofpurification
Marker
Yeast Membrane Proteins Expressed in Yeast
Ynl275w (un-cleaved)
Amount of YNL275W-40
1/6 liter
Comparison of 50 kD-cutoff (expected to retain DDM
micelles) and 100 kD-cutoff (expected to pass DDM
micelles1) membranes in purification of Ste24p (CAAX
protease.)