PURIFICATION OF YEAST MEMBRANE PROTEINS FOR STRUCTURAL GENOMICSCenter for High Throughput Structural Biology

Mark E. Dumont*†, Nadia Fedoriw*, Kathy Clark*, Katrina Robinson*, Gayle Schneider* Michael G. Malkowski‡, George T. DeTitta‡, and Mark Sullivan*

*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

Conclusions1. About 250 yeast transmembrane proteins can be overexpressed to levels of

~ 1mg per liter of culture. The best yields of purified protein are ~0.3 mg/l.

2. Detergents of the fos-choline and maltoside families are generally effective for initial solubilization of many yeast membrane proteins.

3. Efficiency of cleavage of tags on membrane proteins by rhinovirus 3C is much less than for soluble proteins protease and is variable, depending on the target. However intrinsic 3C activity is not inhibited by detergent.

4. IgG binding of ZZ domain tags provides effective purification from whole-cell lysates. Immobilized metal affinity purification is much less efficient.

5. High-purity yeast transmembrane transmembrane proteins are now being produced for crystallization and have successfully served as antigens for generating recombinant single chain antibodies for co-crystallization.

Summary As part of the Center for High-Throughput Structural Biology we are developing technologies for efficient use of yeast for expression and purification of endogenous yeast transmembrane proteins (TMPs) for x-ray crystallography. This combines the benefits of homologous expression of proteins with the advantages of yeast as an organism with well-developed and tractable genetics that is inexpensive to culture. Furthermore, yeast are the only organisms that have been successfully used for heterologous expression of eukaryotic membrane proteins for x-ray crystallography.1,2 We have focused initially on a set of endogenous TMPs that are the highest expressing reading frames in a previously-constructed genomic collection of Saccharomyces cerevisiae expression clones.3 High-expressing TMP targets have been selected based on the availability of established biochemical assays for determining the whether the protein is maintained in a native state. Ligation independent cloning and Gateway cloning procedures have been used to transfer reading frames into vectors that allow galactose-controlled expression of reading frames containing cleavable C-terminal affinity tags. Efficient purification of the solubilized yeast-expressed proteins has been achieved based on affinity chromatography using immobilized metal and IgG affinity matrices with elution by imidazole and by cleavage of tags using rhinovirus 3C protease. However, 3C protease cleavage of target TMPs has been found to be variable and, generally, less efficient than for soluble proteins with similar tags. Yields of purified protein are generally less than 0.3 mg per liter of high-density fermentor-grown culture. Purified proteins and proteins in cell membranes are being used to generate recombinant single chain antibodies to aid in crystallization.1Long SB, Campbell EB, and Mackinnon R.(2005) Science. 309, 897-903.

2Jidenko M, Nielsen RC, Sorensen TL, Moller JV, le Maire M, Nissen P, Jaxel C. (2005) Proc Natl Acad Sci U S A. 102, 11687-91.

3Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, and Grayhack EJ. (2005) Genes Dev. 19, 2816-2826.

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.3 (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 two structures of heterologously expressed eukaryotic

transmembrane proteins have been solved by x-ray crystallography.1,2 Both of these were based on proteins 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 expression

MORF library vector (Gateway cloning)1

pSGP36 (Ligation independent cloning)

PGAL PGK1 5’ LIC site LIC siteORF 3C His10

pSGP38 (Ligation independent cloning)

PGAL PGK1 5’ LIC site LIC siteORF 3C His6ZZ

PGAL ORF 3CHis6ATT siteATT site HA ZZ

Membrane Protein Expression Levels in Yeast

64 -48 -

37 -

26 -

19 -

15 -

6 -

82 -115 -

180 -

IgG LC

IgG HC

Yeast cultures were grown to 15 OD600 then lysed by vortexing with glass beads. Lysates were solubilized in 0.1% Fos-choline 16, bound to IgG sepharose, washed, then eluted in SDS loading buffer containing 9 M urea. Each lane contains the equivalent of 10 mls of the original culture.The indicated molecular weights refer to the un-tagged ORFs. (Tag molecular weight is 18kDa.).

Marker

IgG

alon

e

AA

C1 (34 kD

)

AA

C3 (33 kD

)

CH

O1 (31 kD

)

YN

L275W

(65 kD)

PM

P3 (6 kD

)

RC

E1 (36 kD

)

RH

K1 (53 kD

)

PM

T5 (85 kD

)

3C Protease Cleavage of Target from IgG-Sepharose

Marker

Elu

tion

1

Elu

tion

2

Strip

ped

IgG

1/5 X

18 uL

IgG

Alo

ne

Elu

tion

1

Elu

tion

2

Strip

ped

IgG

1/3 X

Elu

tion

1

Elu

tion

2

Strip

ped

IgG

1/2 X

Elu

tion

1

Elu

tion

2

Strip

ped

IgG

1 X

Elu

tion

1

Elu

tion

2

Strip

ped

IgG

2 XRelative 3C-GST protease added

IgG LC

IgG HC3C-GST

Cleaved YNL275wUncleaved YNL275w

Cleaved tag

21 ug

3C-G

ST

Cultures of a yeast strain expressing YNL275w (putative yeast borate transporter related to mammalian anion transporters) in the MORF library vector were lysed, solubilized in 1% dodecylmaltoside (DDM), then bound to IgG-Sepharose. The bound material was washed in 0.01% DDM, then subjected to cleavage in the presence of various amounts of GST-tagged rhinovirus 3C protease (“Elution” lanes.) Protein remaining on the IgG-Sepharose after cleavage (including IgG) was then eluted in SDS-containing gel-loading buffer (“Stripped.IgG” lanes). Each lane was loaded with the equivalent of 250 ODml of culture .

Advantages of Cleavage of IMAC Affinity TagsImidazole Elutions

AAC1p

3C Cleavage

Marker

Elu

tion

1

Elu

tion

2

Elu

tion

3

Strip

ped

Talo

n

4 g

3C-H

is6

AAC1p

Yeast cells expressing AAC1 (adenine nucleotide transporter) were lysed by vortexing with glass beads, solubilized in 0.5% fos-choline 16, bound to Talon immobilized metal affinity matrix, then washed with buffer containing 0.1% dodecylmaltoside. Elutions were performed by treatment with the indicated concentrations of imidazole (left panel) or by overnight incubation at 4oC with His6-tagged 3C protease (right panel). Each lane was loaded with material derived from 200 ODml of culture.

182 kD -115 kD -

82 kD -64 kD -49 kD -

37 kD -

26 kD -

19 kD -

15 kD -

6 kD -

Marker

5 mM

15 mM

50 mM

150 mM

300 mM

500 mM

-1

0.1 M E

DT

A

+S

DS

500 mM

-2AAC1p + tag

Gel Filtration of Affinity Purified Anion Transporter

-0.0250

-0.0200

-0.0150

-0.0100

-0.0050

0.0000

0.0050

0.0100

0.0150

AU

-100.0

0.0

100.0

200.0

300.0

400.0

500.0

mS/cm

0.0

50.0

100.0 % Buffer B

00:00:00 00:30:00 01:00:00 01:30:00Hr:Min:Sec

1 3 5 7 9 11 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 65 68 71 74 77 80 83 86 89Fractions

1:21:42.4

1:33:07.2

1:46:33.6

171109 79

60

47

35

25

18

14

6

Mar

ker

IgG

Pu

rif.

1st G

el F

ilt.

58 59 60 61 62 63 64 65 66 67 68 69 70

Fractions

YNL275W

3C-GST

71 72 73 74 75 76 77 78 79

Fractions

-0.0250

-0.0200

-0.0150

-0.0100

-0.0050

0.0000

0.0050

0.0100

0.0150

AU

-100.0

0.0

100.0

200.0

300.0

400.0

500.0

mS/cm

0.0

50.0

100.0 % Buffer B

00:00:00 00:30:00 01:00:00 01:30:00Hr:Min:Sec

1 3 5 7 9 11 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 65 68 71 74 77 80 83 86 89Fractions

1:21:42.4

1:33:07.2

1:46:33.6

Abs

orba

nce

(280

nm)

Fractions

Cells expressing the putative anion tranporter encoded by YNL275w from the MORF vector were lysed using an Avestin high pressure microfluidic device, solubilized in 1% dodecylmaltoside, bound to IgG-Sepharose, washed with buffer containing 0.01% dodecylmaltoside, and eluted by overnight incubation at 4oC with GST-tagged 3C protease. The eluate was then subjected to two rounds of gel filtration on Superdex 200.