Short Technical Reports
SHORT TECHNICAL
REPORTS
Manuscripts published in the Short
Technical Reports section are shorter
and less comprehensive in scope than
full Research Reports.
Vectors for Expressing T7
Epitope- and His6
Affinity-Tagged Fusion
Proteins in S. cerevisiae
BioTechniques 24:782-788 (May 1998)
ABSTRACT
We have constructed a series of vectors
(YGALSETs) for the expression of epitopeand affinity-tagged fusion proteins in yeast
cells using the regulated GAL10 promoter.
Fusion proteins produced from YGALSET
plasmids include a leader peptide at the N
terminus that encodes both a T7 gene 10
epitope tag and a His6 affinity tag. The
YGALSET vector series includes centromere plasmids for low-copy plasmid
maintenance and 2 micron episomal plasmids for multicopy plasmid maintenance
and four different selectable markers:
TRP1, URA3, LEU2 and HIS3. We also
provide a convenient approach for transferring cloned genes from a bacterial expression vector into YGALSET vectors by in
vivo recombination and a rapid method to
screen directly for clones that express the
fusion protein of interest.
INTRODUCTION
pRSET vectors (16) are bacterial expression vectors with powerful features. Fusion proteins produced from
the vector include a leader peptide that
encodes an epitope tag and an affinity
tag. The epitope tag encodes the first 12
amino acids of the T7 gene 10 major
capsid protein (T7•Tag) for detection
by immunological techniques. The
affinity tag is composed of six consecutive histidine residues His6 that enable
facile purification of the fusion protein
on metal columns (10) as well as detection of the fusion protein using enzyme-nitrilotriacetic acid-nickel conjugates (1). An enterokinase cleavage site
is located between the leader peptide
and the cloning site so that the epitope
and affinity tags can be excised after
their use, and studies of the nontagged
protein can be performed. Transcription through a polylinker region with
10 or 11 unique sites for cloning the
gene of interest is controlled by the T7
RNA polymerase promoter, which is
tightly regulated and, when induced,
produces high levels of transcript (22).
We wanted to have a similar set of
convenient and versatile features for the
regulated expression of epitope- and
affinity-tagged proteins in the yeast
Saccharomyces cerevisiae. This report
describes a series of yeast expression
vectors (YGALSET) that produce proteins fused to an N-terminal leader peptide that includes the T7 gene 10 epitope tag, the His6 affinity tag and the
enterokinase cleavage site found in the
pRSET bacterial vectors. We describe a
convenient approach for generating
both bacterial and yeast expression
constructs by transferring genes from
pRSET vectors into YGALSET vectors
using in vivo recombination. In addition, we provide a protocol for rapidly
screening yeast transformants directly
for expression of the fusion proteins.
MATERIALS AND METHODS
Strains and DNA Manipulations
E. coli strains XL1-blue (Stratagene,
La Jolla, CA, USA) and MC1066F′ (5)
were used for plasmid propagation.
Plasmid DNA was transformed into
chemically competent E. coli cells (11).
In vivo recombination of plasmids was
performed in transformed yeast (19) or
in chemically competent E. coli cells
(2). Yeast cells were made competent
for DNA transformation with LiCl
treatment (4). Plasmids constructed by
in vivo recombination in yeast were isolated for retransformation into E. coli as
described by Robzyk and Kassir (20).
Plasmid Constructions
The pGAL10 promoter from
pBM272 (13) was excised as an EcoRIBamHI fragment, ends were blunted
with Klenow fragment (New England
Biolabs, Beverly, MA, USA), and the
fragment was inserted into pRSET-C
(16) digested with XbaI and blunted
with Klenow fragment. The resulting
plasmid, pSE285, had the GAL10 promoter driving expression through the
polylinker region. To construct YGALSET351, a BglI fragment including the
GAL10 promoter through the polylinker
Table 1. YGALSET Plasmid Features
Marker
Segregation
Sequence
Reading
Frame*
YGALSET401
TRP1
centromere
B
SacI XhoI PstI PvuII KpnI NcoI HindIII
AF041804
YGALSET352
URA3
centromere
A
SacI XhoI PstI PvuII KpnI HindIII
AF041803
YGALSET351
LEU2
centromere
C
XhoI PstI PvuII KpnI NcoI HindIII
AF041802
YGALSET986
HIS3
centromere
A
SacI XhoI PvuII NcoI
AF041808
YGALSET984
TRP1
2 micron
A
SacI XhoI BglII PstI PvuII KpnI NcoI HindIII
AF041806
YGALSET985
URA3
2 micron
B
SacI XhoI BglII PstI PvuII KpnI HindIII
AF041807
YGALSET983
LEU2
2 micron
A
SacI XhoI BglII PstI PvuII KpnI NcoI HindIII
AF041805
Plasmid
*Open
Unique Cloning Sites
Accession
No.
reading frames corresponding to pRSET A, B or C.
region of pSE285 and YCplac111 [a
LEU2-CEN vector (9)] digested with
HindIII, was co-transformed into
MC1066F′. To select for recombinants,
transformants were plated onto SC
plates lacking leucine (to select for the
LEU2 gene from YCp111 which
complements the leuB mutation in MC1066F′) and containing 5-bromo-4chloro-3-indolyl-β-D-galactopyranoside
(X-gal) (to detect colonies carrying
plasmids with inserts that disrupt the
LacZ gene in YCp111). White Leu+
colonies were screened for the appropriate restriction enzyme digestion pattern.
In an analogous manner, YGAL-
SET352 was constructed from YCplac33 (9) and pSE283 (identical to
pSE285 but in pRSET-A). YGALSET401 was constructed from YCplac22
and pSE284 (identical to pSE285 but in
pRSET-B). YGALSET983, YGALSET984 and YGALSET986 were
obtained by recombination of EcoRVdigested YGALSET352 with PvuII-digested YEplac181, YEplac112 (9) and
pRS313 (21), respectively. YGALSET985 was made from YGALSET401
and YEplac195 (9). All restriction sites
within each polylinker were verified.
Features of the YGALSET plasmids are
summarized in Table 1.
Screening Yeast for Protein
Expression
To screen yeast transformants for recombinants directly by immunoblotting, transformants were first obtained
using the appropriate selection plates
containing glucose (to repress the
GAL10 promoter) and then were grown
overnight in 3 mL of selective liquid
medium containing 2% raffinose + 1%
galactose (to induce the GAL10 promoter). Yeast cells were collected by
centrifugation (1500× g for 5 min) and
washed and then resuspended in 0.3 mL
of extraction buffer (100 mM PIPES at
Figure 1. Use of in vivo recombination to generate fusion proteins in YGALSET vectors using RLF2 as an example. The restriction sites indicated in bold
can be used to liberate genes from pRSET vectors together with the 5′ and 3′ flanking regions that have homology (black) to theYGALSET vectors. To use
YGALSET vectors: (i) clone your favorite gene (RLF2) into the pRSET vector with the appropriate reading frame; (ii) linearize the YGALSET vector at the
XhoI site; (iii) Release RLF2 and the YGALSET homology region (NdeI-BglI) from pRSET-RLF2; (iv) co-transform these linearized fragments into yeast on appropriate selection medium containing glucose; and (v) screen for expression of RLF2 in yeast transformants by growth of the transformants on appropriate selection medium containing galactose. pGAL10:GAL10 promoter; Amp: ampicillin-resistance gene; ORI: ColE1 origin of replication; CEN: centromere.
Short Technical Reports
pH 6.8, 1 mM MgCl2, 1 mM EGTA,
0.5 mM leupeptin, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1% aprotinin). Cells were disrupted by several
rounds of freezing the tube in liquid nitrogen and then thawing the tube at
37°C. Typically, 5% of this extract was
analyzed on immunoblots of sodium
dodecyl sulfate (SDS) polyacrylamide
gels using a 1:3000 dilution of T7•Tag
Antibody (Novagen, Madison, WI,
USA) to detect fusion proteins.
RESULTS AND DISCUSSION
Design and Construction of
YGALSET Plasmids
We used the YCplac and YEplac series of plasmids (9) to provide yeast
replication, selection and segregation
functions for the plasmid backbones.
Each YCplac (low copy) or YEplac
(milticopy) vector included one of three
widely used yeast-selectable markers:
LEU2, URA3 and TRP1. For regulated
gene expression in yeast, we chose the
GAL10 promoter, which is repressed in
cells grown on glucose and induced in
cells grown on galactose (12,13). Tight
repression of the promoter permits the
propagation and conditional expression
of genes encoding toxic proteins.
Cloning Genes into YGALSET
Vectors by Homologous
Recombination
We designed the YGALSET vectors
so that we could easily transfer genes
into the desired YGALSET vector after
cloning them into pRSET-A, -B or -C
(16) with the appropriate reading frame
(Figure 1). This approach exploits the
ability of yeast cells to execute homologous recombination in vivo and minimizes the number of in vitro DNA manipulations necessary to construct
plasmids that can express fusion proteins in both E. coli and yeast cells.
Specifically, the in vivo recombination
approach eliminated the need to construct yeast vectors in three different
open reading frames.
pRSET-A, -B and -C plasmids (16)
differ only in their reading frames starting at the BamHI site in the polylinker.
At the 5′ end of these constructs, there
are 94 bp of homology (between the
NdeI site and the BamHI site of the
polylinker) for recombination that
would yield a fusion protein in the correct reading frame. In vivo recombination upon co-transformation is highly
efficient, typically yielding >95% recombinants (19). In a recent direct immunoblot screen, a cDNA encoding
CHO2 (17) cloned in pRSET was recombined into YGALSET352 and
YGALSET685 (prototype for YGALSET985), and transformant colonies
were screened with T7•Tag antibody;
23 of 28 (ca. 82%) of the transformants
expressed the desired fusion protein.
These plasmids could not be recovered
in E. coli because of their apparent toxicity, illustrating that even constructs
that are difficult to maintain in E. coli
can be directly detected and maintained
in yeast cells.
Functional Complementation from a
YGALSET Vector
The S. cerevisiae gene RLF2 encodes the large subunit of yeast chromatin assembly factor I (15). YGALSET351 carrying the RLF2 gene was
transformed into the S. cerevisiae mutant strain. Expression of the T7-Rlf2p
was detected on Western blots (Figure
2) when cells were grown on galactose.
We did not detect any T7 gene 10
cross-reacting antigens in S. cerevisiae
carrying vector alone (Figure 2). In addition, T7-Rlf2p expression restored
wild-type levels of TEL+CEN antagonism to the rlf2 strain (6) . These results
with the RLF2 gene demonstrate that a
T7•Tag-His6 fusion protein can functionally complement mutant phenotypes in S. cerevisiae rlf2 strains.
Furthermore, the T7•Tag-Rlf2p gene
product was localized by indirect immunofluorescence microscopy (6), indicating that the T7 gene 10 epitope can
be used for immunolocalization in
yeast cells.
In addition to the expression of
RLF2, we have used YGALSET plasmids to express a number of other S.
cerevisiae genomic clones (RAP1,
RLF6, DHH1, SIR4, SIR3, BUD4 and
EST1, unpublished), three mammalian
cDNA clones (Cep135, CHO2 and
hCAFI, unpublished) and a genomic
clone from the hypotrichous ciliate Eu-
plotes crasis (rTBP; Reference 3). In
all of these cases, expression of the fusion proteins was readily detected by
immunoblotting with the T7•Tag antibody and/or with protein-specific antibodies, when they were available. For
the yeast genes, rescue of a mutant phenotype was generally observed, and for
the non-yeast genes, phenotypes attributable to expression of the protein were
observed in all cases. These examples
suggest that the affinity tag plus epitope
tag leader sequence in YGALSET vectors does not interfere with fusion protein function.
Other Applications for YGALSET
Fusion Proteins
Because T7•Tag epitope is readily
detected by indirect immunofluorescence microscopy, YGALSET plasmids are useful for following the localization of tagged gene products. In
addition to localization of Rlf2p (6), we
have detected the distribution of a
T7•Tag-Rap1p fusion protein expressed
in YGALSET351 (pSE408). The
T7•Tag-Rap1p has an identical nuclear
distribution when detected with either
T7•Tag or anti-Rap1p antibodies (S.
Enomoto, unpublished). In all cases we
have tested, T7•Tag fusion proteins expressed from YGALSET plasmids have
yielded clear immunofluorescence signals with little detectable background.
Figure 2. Immunoblot detection of T7•TagRLF2p expression in crude lysates of S. cerevisiae cells. Extracts from cells expressing
T7•Tag-Rlf2 fusion protein (lanes 1 and 3) or
YGALSET351 vector were detected on immunoblots with T7•Tag antibody (lanes 1 and 2) and
were then reprobed with antibody specific for the
endogenous yeast protein Rap1p (lanes 3 and 4).
Short Technical Reports
The His6 affinity tag on the fusion
proteins is useful for isolating the
expressed protein. RLF2p fusion proteins expressed in E. coli from pSE566
or in S. cerevisiae from pME370 have
been specifically enriched on commercially available nickel columns (e.g.,
Ni-NTA agarose [Qiagen, Chatsworth, CA, USA]; data not shown). In
addition, His6 affinity tags have been
used to detect new interacting proteins
and to test physical interactions hypothesized to occur in multimeric protein
complexes (7).
While yeast cell extracts include a
small number of proteins that bind to
metal columns and thus contaminate
fusion protein prepared by a one-step
nickel column affinity protocol (8), the
presence of two different tags in the
YGALSET fusion protein leader peptide should permit a very specific enrichment by performing two sequential
affinity steps: nickel columns and a
T7•Tag antibody column.
Advantages of the YGALSET
Plasmid Series
The YGALSET series of yeast expression vectors drives transcription
from the GAL10 promoter with a multifunctional leader peptide containing the
T7•Tag epitope and a His6 affinity tag.
This leader peptide allows direct
screening for expression of the fusion
protein in yeast using a commercially
available antibody as well as affinity purification of the fusion protein by chromatography on metal affinity resins.
The seven YGALSET vectors include one of four selectable markers
and either a low-copy (CEN) or a multicopy (2 micron) segregation sequence
(Table 1). This set of plasmids, combined with the ability to make appropriate fusion proteins by in vivo recombination with a convenient set of E. coli
vectors (pRSET-A, -B, -C) provides
flexibility and permits the use of many
different applications in a broad range
of yeast strains.
The GAL10 promoter can be tightly
repressed by growth on glucose (14),
an advantage when working with genes
whose products may negatively affect
cell growth or viability. Shifting to
galactose medium results in a high level of gene expression. The 2 micron
YGALSET vectors may be especially
useful for the analysis of dominant negative phenotypes associated with high
levels of gene expression. The shift
from glucose to galactose medium involves a carbon source shift that affects
many metabolic processes in yeast
cells. To avoid such a carbon source
shift, the GAL10 promoter can be induced by the tripartite fusion activator
GAL4.ER.VP16 (18), which will activate expression from the GAL10 promoter when estrogen (provided as βestradiol) is added to the medium.
By using in vivo recombination to
construct YGALSET fusion proteins
from pRSET-based clones, the number
of in vitro manipulations is kept to a
minimum. In addition, the intermediate
pRSET construct containing the gene
Short Technical Reports
of interest is useful for high levels of
expression from the T7 promoter in E.
coli. Comparison of the same fusion
protein produced in E. coli and S. cerevisiae may reveal differences in the
posttranscriptional modifications of the
protein produced in prokaryotic vs. eukaryotic cells. In vitro cloning is also
simplified because all of the polylinker
sites in the pRSET plasmids are available for cloning. While direct cloning
into YGALSET plasmids is certainly
possible, only a subset of the YGALSET polylinker sites are unique in these
larger plasmids, and only a subset of
the 3 reading frames required for this
approach are present in the available
YGALSET plasmids.
A minor limitation of YGALSET
plasmids is that XbaI and NdeI are the
only two restriction sites available for
cleavage 5′ to the insert sequence in
pRSET vectors. If these sites are pre-
sent in the gene to be expressed, PCR
amplification using primers near the
XbaI-NdeI and BglI-PvuI sites can be
used to generate the insert fragment for
co-transformation with a YGALSET
vector.
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We thank Deborah Carlson, Carolyn
Price, Jodi Lew and Ryoko Kuriyama for
sharing results prior to publication, Vitaly
Shapolov for patient technical help and
Paul McCune-Zierath for technical assistance. This work was supported by the National Institutes of Health (Grant No.
GM38626). Address correspondence to Judith Berman, Department of Plant Biology,
University of Minnesota, 220 Biological
Sciences Center, St. Paul, MN 55108, USA.
Internet:[email protected]
Received 18 September 1997; accepted 12 January 1998.
Shinichiro Enomoto,
Guanghui Chen and
Judith Berman
University of Minnesota
St. Paul, MN, USA