Transcript Document

Expression and Purification of Integral Membrane Proteins from Yeast for the
Center for High-Throughput Structural Biology
*
Clark ,
*
Fedoriw ,
*
Randles ,
Kathleen
Nadia
Joan
Mary Rosenblum‡,
‡
‡
†*
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
Abstract
Progress Summary
1. Protein expression –
•
High-purity yeast transmembrane proteins are now being produced for crystallization. Utilizing the
ADH2 promoter over the GAL1 promoter has increased the protein yield of Bor1p by 2X and
reduced cost of expression by eliminating galactose as inducer. Expression testing of other proteins
using this promoter are in progress. The best yields of purified membrane protein are 0.6 mg/l of
culture.
•
To eliminate the problem of expression plasmid loss during growth in both rich and selective media,
we are testing an integrating vector for stable integration into the dispersed Ty δ sites of S.
cerevisiase as in Parekh et. al. (1996).
•
To further reduce protease activity in the yeast lysates, we are testing an additional protease
deficient strain BJ1268 (pep4-, prb1-, plus prc1- deficient). Also, we have determined that the best
protease inhibitors for yeast proteins are PMSF and chymostatin over Peflaboc SC™ (Roche).
SEC with LS, UV, RI Detection
Blue Native Gel
(Sample required >10 ug protein)
( >5 ug protein)
may interfere with crystal contacts formation during
crystallography.
• Removal of detergent may cause proteins to aggregate.
300
LS signal
A
UV signal
RI signal
250
Before detergent reduction
200
Bor1p
monomer
150
Detergent
micelle peak
2. Protein/Detergent AnalysisTechniques for monitoring protein oligomerization, eg. SEC with LS,UV, and RI and blue native
electrophoresis, have been implemented and are being used to monitor protein oligomerization
during detergent removal and after adding ligands, inhibitors, and other additivies.
1048
720
0
6
7
8
9
10
11
12
13
14
15
16
Time (min)
480
-50
300
Colorimetric detergent assay has been implemented for analysis of DDM levels in protein samples.
SEC with three detector system is being developed for detergent analysis.
B
UV signal
RI signal
242
250
Challenges
The goal of “E. coli-fying” yeast as an expression system for membrane proteins will benefit
from ongoing development in the following areas:
•
Increasing native protein production by continued development of culture and induction conditions.
Optimization of detergent selection for solubilization, purification, and crystallization of proteins.
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 a inexpensive high-affinity resin for affinity purifications.
Expansion of expression and purification techniques to include a wider range of targets refractory to
current methods.
Bor1p (uncleaved)
GAL1 promoter - Induction with
galactose after glucose depletion
ZZ His10
0.080
12*
Bor1p
IgG
For Bor1p under the GAL1 promoter optimal
expression occurs at around 12. hrs then
decreases. For the ADH2 promoter optimal
expression is reached at around 20 hrs.
when cell densities are higher.
ADH2 Promoter
Elutions after GST resin
IgG Elution 3
IgG Elution 2
IgG Elution 1
8
9
10
20X
30X
0.020
0.010
0.0
-0.010
-100.0
00:00:00
01:00:00
50X
0.01
0.005
720
15
B 10X
480
UV signal
RI signal
242
After detergent reduction
146
66
6
20X
30X
40X
7
8
9
10
filtrate
Panel A:
purified
protein
using repeated cycles of dilution and concentration (Millipore
50 kDa concentrator).DDM was analyzed using a colorimetric
method (Urbani and Warne, 2004). The DDM concentration of the
diluting buffer is 10X lower than the critical micelle concentration
(buffer DDM = 0.001%).
40X
50X
12
13
14
15
Time (min)
Blue native gel for
Panel D
F
250
0.6
1048
720
480
LS signal
UV signal
RI signal
200
0.4
116 kDa
Bor1p in
C12E8 (Panel
D)
214 kDa
397 kDa
658 kDa
65 kDa
62 kDa
120 kDa
185 kDa
130 kDa
146 kDa
250 kDa
390 kDa
Bor1p in
C12E8
(Panel E)
320 kDa
124 kDa
150 and 300
kDa
Molecules
Bound
Bound
Total
Detergent:Protein Detergent Detergent Detergent
(Ratio:
Colorimetric SEC w/ Colorimetric
mole:mole)
Assay1
UV,RI,LS2
Assay3
DDM:Bor1p
145:1
149:1
228:1
(Panel B)
C12E8:Bor1p
-- 4
101:1
-- 4
(Panel D)
1. Calculated based on DDM concentrations determined by
colorimetric analysis. (DDM concentration in peak maximumDDM concentration at peak minimum).
2. Calculated using three detector method.
300
Bor1p dimer
242
3. Calculated based DDM concentration determined by colorimetric
analysis of purified protein and total protein concentration.
4. No detergent assay available for C12E8.
150
146
66
100
0.2
50
0
Concentrator Filtrate Dilutions
Panel B: Using SEC with UV, RI, LS detection (see right panel),
detergent levels can be evaluated based on the ratio of micelle
peak to protein peak area. Note the increase of the area ratio at
dilution 5, indicating protein loss at lower DDM concentrations.
This SEC method can be a sensitive indicator of protein stability.
Panel C: SDS Page analysis of Bor1p at the different
stages of detergent removal.
Bor1p
11
SEC of Bor1p in C12E8 detergent. Panel C: Bor1p
before detergent removal. Panel D: Bor1p after
detergent removal. Note increase in protein
oligomerization and loss of micelle peak.
50X
Bor1p in
DDM
(Panel B)
Calculation of Detergent Bound to Protein
10
RI Analysis of Bor1p Concentrates
0.8
Blue Native
Gel
60
02:00:00
Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5
conc
conc
conc
conc
conc
Concentrator Filtrate Dilutions
Dilution 5
30X
14
110
Hr:Min:Sec
0.015
Dilution 10X 20X
13
LS signal
160
100.0
0.000 0.0
40X
12
D
0.030
Detergent Analysis of Bor1p
Concentrator Filtrates
A
11
200.0
Dilution 1,2
Dilution 3
Dilution 4
filtrate
filtrate
Detergent
(DDM) isfiltrate
removed from
the
*300 OD mls
7
210
0.050 50.0
0.040
Accurate Molecular Mass
Determination using Static Light Scattering
Bor1 MW: monomer 65 kDa, dimer = 130 kDa, trimer = 195 kDa
6
300.0
Blue Native Electrophoresis (NativePAGE) can also be
used to monitor protein oligomerization. Coomassie dye replaces the detergent around the protein preventing aggregation
during electrophoresis. The MWs obtained are not accurate but can
be calibrated against other methods. (Heuberger, et. al., 2002).
SEC (UV
detector
only)
SEC
(w RI, LS,
UV
detection)
80
400.0
Ynl275w
Size Exclusion Chromatography with detection by UV
absorbance (UV), refractive index (RI), and static light
scattering (LS) detection can be used to monitor protein
oligomerization. With knowledge of protein extinction coefficients
and the instrument calibration constant, the protein mass can be
determined independent of hydrodynamic radius and without
interference from protein posttranslational modifications, eg.
glycosylation, and bound detergent. Additionally, the method can be
used to estimate the amount of detergent bound to the protein.
(Folta-Stogniew, 2006).
Method
Micelle peak
Determining and Reducing Detergent Concentrations
In Bor1p Purifications
0.02
NativePAGE Gel for
Panel B
LS signal
UV signal
RI signal
C
-40
0
Gal1 promoter
The ADH2 promoter is a glucose
repressible promoter (Price, 1997). For
expression, the yeast cells are grown in
rich media. Expression is initiated, after
glucose is consumed.
Induction Time (hrs)
0
6*
24
30
0.060
psi
% Dodecylmaltoside
Detergent
Using the ADH2 Promoter Increases Protein Expression for Bor1p
IgG stripped urea/SDS
protease-deficient yeast strains.
2. Cell lysis by Avestin C3 homogenizer.
3. Membrane pellet isolation and solubilization in detergent.
4. Affinity purification by IMAC and/or IgG resins and 3C rhinovirus protease cleavage from
resins.
5. Size exclusion chromatography (SEC) purification to remove aggregates.
6. Concentration of protein with detergent removal by cycles of dilution and concentration.
IgG super rebound to IgG
1. Yeast growth during expression in YPD with autoinduction (GAL1 or ADH2 promoter) in
Urea/SDS stripped Talon
Talon Elution 3
Purification Strategy
Talon Elution 2
Talon Elution 1
0.070
Gel Filtration
Superdex 200
16
mV
LIC site 3C
15
-20
mV
ORF
14
30
3C-GST 5 g
PGK1 5’ LIC site
13
130
2 6 10 16 22 28 34 40 46 52 58 64 70 76 82 88 94 100107114121128135142149156163170
0.100
500.0
100.0
% Buffer B
0.090
ADH2 promoter - Glucose
repressible promoter
12
Before detergent reduction
Fraction
#
Fractions
Bor1p 5 l
pSGP46 (Ligation independent cloning)
PAHD2
3C-GST
ZZ His10
Bor1p 10 l
LIC site 3C
11
1236
1048
Marker
ORF
2:26:46.4
PGK1 5’ LIC site
10
SEC of Bor1p in DDM detergent.Panel A:
Bor1p before detergent removal. Panel B: Bor1p
after detergent removal. In DDM Bor1p is a
monomer. Note change in area of micelle peak.
1:23:12.0
PGAL
9
3-12% gel
Detergent: dodecyl maltoside (DDM)
Culture: 96,000 ODmls
1:38:14.4
pSGP40 (Ligation independent cloning)
Bor1p
(cleaved)
8
Time (min)
Multi-step purification of the S. cerevisiae
boron transporter Bor1p
1:14:08.0
LIC site 3C His10
7
-50
180
Ratio RI Signal
Area micelle/Area protein
ORF
6
mV
•
•
0
0.5 l
•
•
MW
50
pSGP36 (Ligation independent cloning)
PGK1 5’ LIC site
Reduced
detergent 20
micelle peak
100
Vectors for yeast membrane protein expression
PGAL
150
mV
Yeast Membrane Proteins Expressed in Yeast
146
66
200
1.0 l
•
LS signal
Bor1
monomer
•
50
After detergent reduction
1. To date, eight structures of different heterologously expressed eukaryotic transmembrane proteins
have been solved by x-ray crystallography. Five 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.
4-16% Gel
100
•
Protein/Detergent Interactions
• Excess detergent in membrane protein preparations
mV
Compared to the large amount of structural information available for soluble proteins,
transmembrane proteins (TMPs), and particularly TMPs from eukaryotes, remain poorly
characterized, despite their physiological and medical importance. The Center for High-Throughput
Structural Biology is working to develop improved protocols for expression and purification of TMPs
in the yeast Sacchromyces cerevisiae. We have focused on a set of the highest-expressing
endogenous yeast TMPs for which there are established biochemical assays for determining whether
the protein is maintained in a native state. Initial purifications were based on expression of the genes
under control of a galactose inducible promoter, but higher cell densities and improved expression
have recently been obtained through use of the yeast ADH2 promoter. Wide variations have been
observed in the behavior of different TMP targets during purification- some can be readily purified,
while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or
remain tightly associated with the matrices even after cleavage of the affinity tags. We have taken
several steps that have effectively reduced proteolysis by endogenous yeast proteases. Size
exclusion chromatography in conjunction with static light scattering, refractive index detection, and
UV absorption, is being used to characterize the oligomeric state, heterogeneity, and amount of
bound detergent in purified protein preparations. The results of these analyses are also being
compared with native gel electrophoresis and with colorimetric assays for determining detergent
concentrations. Approaches that we have developed for exchanging from an initial detergent that
efficiently solubilizes TMPs to alternative detergents more amenable to crystallizations, as well as
procedures for efficiently removing excess detergent from purified protein-detergent complexes,
appear to be important for successful crystallization.
Methods for Evaluating Membrane Protein Oligomerization
0
6
-50
7
8
9
10
11
Time (min)
12
13
14
15
SEC of Bor1p in C12E8 detergent bound to inhibitor.
In C12E8 Bor1p is a preferentially a dimer. Compare
the oligomerization state of Bor1p in panels C and D
with F. SEC is a fast method for evaluating the
oligomerization of proteins.
20
Blue native gel for
Panel F
Crystals from Bor1p purified Crystals purified in C12E8
in DDM
with anion channel inhibitor