Expression and Purification of Integral Membrane Proteins from Yeast for the Center for High-Throughput Structural Biology

Kathy Clark*, Nadia Fedoriw*, Katrina Robinson*, Mark Sullivan†, 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

Current bottlenecks/solutions1. High-purity yeast transmembrane proteins are now being produced for crystallization and have

successfully served as antigens for generating recombinant single chain antibodies for co-crystallization. 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.

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 C-terminal 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 Strategies30 Target ORFs are currently selected based on the following criteria:

1. Prediction of two or more transmembrane segments based on TMHMM and HMMTop

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)

4. Existence of a published procedure for assaying native state of produced protein.

Yeast Membrane Proteins Expressed in Yeast1. 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, post-translational modifications, intracellular trafficking

5. Rapid and inexpensive conditions for culturing yeast cells

Vectors for yeast membrane protein expressionMORF library vector (Gateway cloning)1

pSGP36 (Ligation independent cloning)

PGAL PGK1 5’ LIC site LIC siteORF 3C His10

pSGP40 (Ligation independent cloning)

PGAL PGK1 5’ LIC site LIC siteORF 3C His10ZZ

PGAL ORF 3CHis6ATT siteATT site HA ZZ

Fermentor culture(autoinduction galactose)

Lysate

Sup

Bind to IMAC or IgG affinity matrices

Harvest, lyse (Avestin)

100,000 x g spin

Gel filtration

Concentrate

Pellet

3,000 x g spin

Sup Membrane Pellet1.2M KCl;

120,000 x g spin

Detergent solubilization26,000 x g spin

Pellet (solubilized protein)

Static Light Scattering

Crystallization trials

ORF cloning

Target selection

Salt-washed membranes

3C protease cleavage

Imidazole elution

Detergent exchangeand dilution

Culture conditions: IssuesS. 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.

Talon-binding proteases of yeast

Ste24p cleaved from Talonwith GST-tagged 3C protease

Ste24p stripped from Talon using EDTA

Strain 1: BJ5460 pep4- prb1 Strain 2: EJG1117 pep4- prb1-

Strain 3: EJG1364 pep4- PRB1+

Each purification:300 OD mls

Ste24-40uncleavedSte24-40

cleaved

3C-GST

PMSF + - + - + - + - + - + -

1 2 3 1 2 3Strain

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.)

Mar

kers

0.1 M NH4Br 0.1M Tris pH 8

20% PEG 80000.1 M NH4Br 0.1M Acetate pH 5

20% PEG 80000.2 M KSCN pH 7

20% PEG 3350

Anion transporter YNL275w (pSGP40, cleaved)

KCl-stripping of membranes

27

5W

-40

from

IgG

Ma

rke

r, 15

u

L

Wa

sh

1

Wa

sh

2

5 m

M im

ida

zole

15

mM

imid

azo

le

50

mM

imid

azo

le

15

0 m

M im

ida

zole

30

0 m

M im

ida

zole

50

0 m

M im

ida

zole

ED

TA

-Strip

pe

d

Amount of YNL275W-40 1/6 liter

Loading: 1/200th ofpurification

27

5W

-40

, from

IgG

Ma

rke

r

5 m

M im

ida

zole

15

mM

imid

azo

le

50

mM

imid

azo

le

15

0 m

M im

ida

zole

30

0 m

M im

ida

zole

50

0 m

M im

ida

zole

ED

TA

-Strip

pe

d

YNL275w-40

Un-stripped membranes 0.7 M KCl-stripped membranes

Mar

ker

50 k

Da

con

cen

trat

e

50 k

Da

filt

rate

100

kDa

con

cen

trat

e

100

kDa

filt

rate

Concentration of purified protein in the presence of detergent

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.)

Mar

ker

3C-H

is6

elu

tion

3C-H

is6

elu

tion

500

mM

imid

azol

eel

utio

n

20

ul

10 u

l

5 ul

3C-6

HIS

pro

teas

e(

7 ug

)

3C-G

ST

pro

teas

e(

5 ug

)

49 kDa

Purification from96,000 OD mls

1-Step Purification of Ste24p (CAAX

protease) on Talon Ste24p expressed from vector pSGP40 was solubilized from KCl-washed membranes, bound to Talon, then eluted by cleavage with His6-tagged 3C protease. After elution, the Talon column was treated ith 500 mM imidazole to visualize

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

psi

-100.0

0.0

100.0

200.0

300.0

400.0

500.0

0.0

50.0

100.0 % Buffer B

00:00:00 01:00:00 02:00:00Hr:Min:Sec

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 170Fractions

1:14:08.0

1:23:12.0

1:38:14.4

2:26:46.4

Fraction #

Gel Filtration Superdex 200

Ynl275w

Multi-step purification of the anion transporter YNL275w

Ta

lon

Elu

tion

1

Ta

lon

Elu

tion

2

Ta

lon

Elu

tion

3

Ure

a/S

DS

str

ipp

ed

Ta

lon

IgG

su

pe

r re

bo

un

d t

o I

gG

IgG

Elu

tion

1

IgG

Elu

tion

2

IgG

Elu

tion

3

IgG

str

ipp

ed

ure

a/S

DS

Elu

tion

s a

fte

r G

ST

re

sin

Ynl275w(cleaved)

3C-GST

Ynl275w (un-cleaved)

Mar

ker

Yln

275

w 1

0 l

Ynl

275

w 5

l

3C-G

ST

5

g

Detergent: dodecyl maltosideCulture: 96,000 ODmls The C-terminal tags of many yeast membrane

proteins may be obscured by detergents1. Many tagged yeast membrane proteins are not efficiently cleaved by 3C protease

2. The activity of 3C protease is not intrinsically sensitive to detergents.

3. Inefficient cleavage can sometimes be overcome by adding large amounts of protease.

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.)

5. Also: Use of Nickel-NTA resin inhibits subsequent 3C protease cleavage whereas use of cobalt (Talon) does not.

                                                  

tag (Z-domain)

protein

detergent

3C-cleavage site

His-His-His-His-His-His