PURIFICATION OF YEAST MEMBRANE PROTEINS FOR STRUCTURAL GENOMICS

1

PURIFICATION OF YEAST MEMBRANE PROTEINS FOR
STRUCTURAL GENOMICS Center 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

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-controlle
d 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.
Advantages of Cleavage of IMAC Affinity Tags
Imidazole Elutions
3C Cleavage
182 kD - 115 kD - 82 kD - 64 kD - 49 kD - 37 kD
- 26 kD - 19 kD - 15 kD - 6 kD -
AAC1p tag
AAC1p
AAC1p
5 mM
0.1 M EDTA SDS
Marker
15 mM
50 mM
150 mM
300 mM
500 mM-1
500 mM-2
Marker
Elution 1
Elution 2
Elution 3
Stripped Talon
4 ?g 3C-His6
Membrane Protein Expression Levels in Yeast
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 OD?ml of culture.
180 -
115 -
82 -
64 - 48 -
IgG HC
Yeast Membrane Proteins Expressed in Yeast 1. 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
37 -
IgG LC
26 -
19 -
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.).
15 -
6 -
PMT5 (85 kD)
3C Protease Cleavage of Target from IgG-Sepharose
Uncleaved YNL275w
Targeting Strategies 30 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.
Cleaved YNL275w
IgG HC
3C-GST
Conclusions 1. 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.
IgG LC
Cleaved tag
Elution 1
Elution 2
Elution 1
Elution 2
Elution 1
Elution 2
Elution 1
Elution 2
Elution 1
Elution 2
Stripped IgG
Stripped IgG
Stripped IgG
Stripped IgG
Stripped IgG
Marker
21 ug 3C-GST
18 uL IgG Alone
Relative 3C-GST protease added
1/5 X
1/3 X
1/2 X
1 X
2 X
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 OD?ml
of culture .