Gene. 104 (1991)

107-I 11

Q 1991 Elsevier

Science

GENE

Publishers

B.V. All rights reserved

107

0378-I 119/91/$03.50

05055

Improved shuttle vectors for cloning and high-level Cu2+ -mediated expression of foreign genes in yeast (Recombinant heterologous

DNA; Saccharomyces gene expression)

Ian G. Macreadie, CSIRO

cerevisiae;

Ourania Horaitis,

Division of Biomolecular

CUP1 ; Trichostrongylus

colubriformis;

helminth;

copper-ion

regulated;

Amanda J. Verkuylen and Keith W. Savin *

Engineering,

Parkville, Victoria 30.52 (Australia)

Received by P.A. Manning: 16 July 1990 Revised: 19 February 1991 Accepted: 1 March 1991

SUMMARY

New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described. One vector, pYEULlacZ, is based on pUCl9 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by IacZ inactivation. In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains. The presence of the leu2-d gene appears to promote replication at high copy numbers. The introduction of CUP1 cassettes allows these plasmids to direct Cu2+ -regulated production of foreign proteins

in yeast. We show the production

of a helminth

INTRODUCTION

The cloning of foreign genes in yeast often involves the use of shuttle vectors with ori’s and selectable markers for Correspondence to: Dr. LG. Macreadie, Engineering,

343 Royal Parade,

Tel. (+ 61-3)3424200; * Present Victoria

address:

CSIRO,

Parkville,

Division of Biomolecular

Victoria

3052 (Australia)

Fax (+ 61-3)3475481. Calgene Pacific Pty. Ltd., 16 Gipps St., Collingwood,

3066, (Australia)

Tel. (+ 61-3)4199302;

Fax (+ 61-3)416

Abbreviations:

Ap,

captoethanol;

CUPI,

ampicillin; yeast

1761.

/?Gal,

gene

j?-galactosidase;

encoding

CUPlE, alleles of CUP1 utilized for expression deoxyribonucleoside

triphosphate;

excretory/secretory

30-kDa

propyl-malate

dehydrogenase;

&2-d,

CUPlB,

of foreign genes; dNTP,

kb, kilobase

glycoprotein;

j?ME, B-mer-

metallothionein;

or 1000 bp; ESgp30,

LEU2,

LEU2

gene

encoding

with a deleted

iso-

promoter;

MCS, multiple cloning site; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ori, origin of DNA replication; p, plasmid; PAGE, polyacrylamide-gel

electrophoresis;

myces; SDS, sodium Na, citrate pH 7.6; decarboxylase; extract;

R, resistant/resistance;

dodecyl sulfate; SSC, URA3, gene encoding

wt, wild type; YEPD, 2% glucose/2%

[ 1, denotes plasmid-carrier

state.

S, Saccharo-

0.15 M NaCl/O.OlS M orotidine-5-phosphate peptone/l%

yeast

antigen

as an example

of the vector application.

both E. coli and S. cerevisiae. Such vectors include the yeast episomal (YE) plasmids (reviewed by Rose and Broach, 1990), which replicate to medium or high copy number, utilizing yeast replicons from the native yeast 2 pm circle plasmid. Selection of these plasmids in yeast is usually achieved by complementation of an auxotrophic mutations (e.g., feu2, ura3, trpl, his3, etc.). The availability of practically non-reverting ura3 and leu2 host strains has meant the frequent use of vectors having either URA3 or LEU2 as selectable markers in yeast. Useful examples are those vectors derived by Gietz and Sugino (1988). By in vitro mutagenesis they produced new alleles of the LEU2 and URA3 yeast genes, removing many 6-bp restriction sites without changing the translation products. They utilized these alleles in yeast-E. co/i new shuttle vectors with a pUC1P derived MCS and the ability to screen for E. coli recombinants by a j?Gal color assay. We have made further improvements to the utility of yeast shuttle vectors. To provide a choice of selectable markers, we placed both the LEU2 and URA3 yeast genes on one plasmid, pYEULlacZ, employing the leu2-d gene instead of the wt LEU2 gene to increase the copy number

108 of the vector in yeast. The use of leu2-d to increase

vector

EXPERIMENTAL

copy number was recently reviewed by Rose and Broach (1990). A useful advantage of the high copy number is high expression levels (Macreadie et al., 1989). We have added the Cu* + -inducible expression cassettes, CUPlB and

AND DISCUSSION

(a) Construction of pYEULlacZ The strategy for constructing pYEULlacZ was to produce a vector with the properties of YEplac 195, the URA3containing vector of Gietz and Sugino (1988), but with the additional properties of leu2-d selection and amplification.

CUPIE (Macreadie et al., 1989), to pYEULlacZ to construct new vectors, pYEULCB and pYEULCE for highlevel Cu2+-inducible expression of gene fusions. As an example we show the Cu* + -induced production in yeast of a protein normally secreted by the intestinal parasitic nematode Trichostrongylus colubriformis.

Installing the leu2-d gene into YEplac195 was achieved by a three-step cloning strategy as described in Fig. 1. The first step involved the construction of pYILC5, a vector containing leu2-d, constructed from pYELC5 to have

NaiI+Hindmd~geot, blunt r*-

with T4 PO,.,

ligate.

1

Reolace LEUZ

tra9mant

0.6kb

tram

Clone

Clrl-EcoRv

I

VEplaClSI

HpaI-Slut site

of

IleuZ-d) into

fragment

Hp.3 I

VEplacl95

inas,

k

=

Fig. 1. Construction

of pYEULlacZ.

Vectors

used in the construction

of pYEULlacZ

are shown

pVELC5

B

VEplac

181

zziz===

VEplac

195

mm

Yeast

ori

mIL$

vaast

g*nc

-

gacterlal

OrI

ZZZ$

Bacterial

gene

along with their unique

restriction

sites; some other

relevant restriction sites are shown in parentheses. Bacterial and yeast ori’s are denoted as oriB and oriY, respectively. ApR encodes B-lactamase for selection of plasmids, while IucZ contains the pUCl9 MCS and enables blue/white color selection of recombinants in E. cob. Selection of plasmids in yeast utilizes complementation

with UR43, LEU2 or leu2-d, a version of LEU2 that has a deleted promoter.

can be used for Cu* + -regulated

expression

by deleting the 1.2-kb HindIII-NsiI fragment, KpnI sites in the leu2-d gene, accomplished YEplacl81 fragment.

(Gietz and Sugino, This HpaI-SruI

of foreign genes in yeast. The removal producing a vector designated by replacing the &I-EcoRV

1988). The replacement

fragment

was ligated

created

a new vector,

of the unwanted

CUPlB

encodes

CIuI site in pYELC5

yeast metallothionein

(Macreadie,

and

1990) was achieved

as pYILC5. The second cloning step involved the removal of the EcoRI and fragment of pYILC5 with the corresponding ClaI-EcoRV fragment from pYILCSN,

into the HpaI site of YEplacl95

(Gietz

from which the desired leu2-d gene was isolated and Sugino,

1988).

as a HpaI-Srul

109 unique ClaI and EcoRV sites (first step in Fig. 1). The presence of EcoRI and KpnI sites in leu2-d was undesirable and their removal was accomplished by replacement of the internal ClaI-EcoRV fragment, containing both EcoRI and KpnI sites, with the corresponding fragment from the LEU2 allele constructed by Gietz and Sugino (1988) to create the vector pYILC5N (second step in Fig. 1). The final step in the construction of pYEULlacZ involved the transfer of the modified /eu2-d fragment from pYILCSN, as a HpaI-StuI fragment,

to YEplacl95.

TABLE Mitotic

II stability

Plasmid il

pYELC5

in transfor-

mation (see Table I) by following the procedures of Gietz and Sugino (1988). The Ura + transformation frequency of pYEULlacZ is comparable to the transformation frequencies previously obtained by Gietz and Sugino (1988) with their vectors; however, the Leu + transformation is just frequency. The Leu + 4% of the Ura+ transformation transformation frequency of pYELC5 is also quite low and these data are in accord with our previous observations that the leu2-d gene gives a lower transformation frequency when selection is on medium lacking leucine (I.G.M. and P.R. Vaughan, unpublished).

(c) Stability and copy number of pYEULlacZ The mitotic stability of the plasmids in yeast strain DBY747 was monitored following zero, ten and thirty generations of growth in rich media (see Table II). While pYELC5 transformants are extremely stable, those containing pYEULlacZ are less stable, but slightly more stable than the YEplac181 transformants. These data suggest that pYEULlacZ and YEplac 18 1 replicate to a lower copy number than pYELC5. Data on relative plasmid copy number was obtained by analysis of DNA in transformants grown in selective condi-

TABLE

I

Transformation

of yeast a

Plasmid b

Transformation

frequency’

Ura + selection pYELC5 pYEULlac2 a Strain DBY747

Leu + selection

0

10

4700

170

(a hisj7d-1 Ied-3,112 ~~1-289 ~13-52)

as host for transformations. ’ Plasmids are described in Fig. 1. pYEULlacZ

was employed

transformants

were both

Ura + and Leu +

c Transformations

were performed

according

to the method of Gietz and

Sugino (1988). Frequencies are the average obtained mations and represent the number of transformants

from five transforper pg DNA.

after growth

in non-selective

media’

0 generation

10 generations

30 generations

>99% 96%

98% 75%

947,

YEplaclSl pYEULlacZ

99%

81%

73”;

a Plasmids

are described

footnote

(b) Transformation with pYEULlacZ We examined the properties of pYEULlacZ

Retention

in Fig. 1. Host was the same as in Table I,

a.

’ Transformants

were grown in liquid YEPD

onto YEPD-agar

after the number

were then replica-plated tryptophan,

60%

histidine

which retain the Leu’

medium

of generations

and then plated

indicated.

to minimal medium supplemented and uracil

to determine

the number

Colonies

with 20 pg/ml of colonies

phenotype.

tions. Aliquots of serially diluted total DNA, prepared according to Davis et al. (1980) were slot blotted onto nitrocellulose and probed separately with the oligo 5’-GCTAGAGTAAGTAGTT (complementary to the ApR gene) or DBY747 DNA. Hybridization of the oligo was at room temperature in 10 x Denhardt’s solution (Denhardt, 1966) 6 x SSC, followed by washes in 3 x SSC at 37°C. Hybridization with DBY747 DNA was under stringent conditions recommended by Sambrook et al. (1989). After hybridization the filters were washed and autoradiographed. Autoradiographs (not shown) were scanned with a Molecular Dynamics (Sunnyvale, CA) computing densitometer to estimate the relative autoradiographic density. Calculations indicate that pYELC5 and pYEULlacZ (from cells grown with or without leucine) replicate to a level 12 times and 6 times higher, respectively, than YEplac 18 1. The elevated copy number of both pYELC5 and pYEULlacZ is expected in selective medium; however, the observation that these levels remain high even in the presence of leucine is as yet unexplained. Baldari et al. (1987) have also observed this phenomenon. (d) Expression vectors derived from pYEULlacZ New Cu2 + -inducible expression vectors were constructed based on the expression cassettes, CUPlB and CUPlE (Macreadie et al., 1989) and pYEULlacZ. These vectors, designated pYEULCB and pYEULCE, respectively, and illustrated in Fig. 2, were constructed by cloning the cassettes as SacI(T4 DNA polymerase treated)-toHind111 fragments. The cassettes were cloned into pYEULlacZ which had been digested with NruI, then treated with T4 DNA polymerase in the presence of dNTPs and finally cut with HindIII. The resulting constructs have four unique restriction sites (EcoRI, SalI, PstI and BamHI) available for the cloning of genes under control of the Cu* + regulated promoter. We have examined the ability of pYEULCE to direct the

110

Hindu

synthesis of a foreign protein in yeast. A 0.5kb cDNA fragment encoding ESgp30, a secreted glycoprotein from the parasitic nematode Trichostrongylus colubriformis (Savin et al., 1990) was cloned into the EcoRI site of pYEULCE, fusing the reading frame of ESgp30 with that of the CUPlE gene. The cDNA fragment lacks an initiating ATG but retains a translation terminator and polyA-addition signal. The resulting plasmid, pYEULCE/30B/7, was transformed

:

ApR I

f

into yeast strain DBY747 and subjected to the Cu’ ’ -induction protocol described in Fig. 3. Lysates of Cu2 ’ -induced cell cultures were examined by SDS-PAGE (Laemmli, 1970) and Western blotting (Towbin et al., 1979). As can be seen in Fig. 3, a basal level of production of a 1%kDa protein is detected by the anti-ESgp30 serum (Savin et al., 1990) without Cu 2+ induction (zero time). This protein production increases dramatically within the first 30 min after induction and continues to increase, albeit at a much slower rate, up to at least 24 h as seen in Coomassie bluestained gels and Western blots of whole cell lysates. The level of this 15kDa protein, after 24 h in the presence of CL? + , comprised 13 “/, ofthe total cellular protein, as judged by computing laser densitometry of Coomassie blue-stained

AatlI 5 URA3

\ Nde EcoRV

1

NcoI

Fig. 2. The pYEULCB restriction

expression

sites in the reverse

orientation.

(and reversed

CUP1E) cassettes

et al. (1991).

Annotations

upstream

activation

arrowhead,

vector.

sites are shown. pYEULCE

stop codon.

M

The complete

sequence

are

as follows:

tilled box, MCS;

Other

details

0

-5

V

and other

relevant

SalI, PstI and EcoRI of the CUPIB

(open boxes) can be found in Macreadie

in CUPIB

sequences;

Umque

has BumHI,

hatched

box,

line, the startcodon;

are as in Fig. 1.

1

2

10

24

h

kDa

M

kDa

130 75

V

0

-5

1

2

10

24

h

130 75

50

50

39

39

27

27

17

B y *+ -inducible synthesis of parasite antigen. Fig. 3. Cu synthetic medium containing histidine and tryptophan adding

CuSO,

(Seikagaku

to 0.5 mM. Cell cultures

Kogyo,

Tokyo)

to remove

were grown

A yeast transformant

containing

the plasmid

pYEULCE/30B/7

was grown

for 18 h in minimal

(20 pg/ml). The cells were then harvested

and grown for a further 2 h in the same medium before and then collected, treated with zymolyase for 0, 0.5, 1, 2, 10 and 24 h in the presence of Cu”

the cell wall (Jagadish

et al., 1990) and lysed by the addition

of an equal volume of SDS-PAGE

sample buffer

(125 mM Tris. HCI pH 6.8/10x Fico11/2.5% fiME/Z% SDS/O.l% bromophenol blue). Aliquots ofcelllysates were analysed by 0.1% SDS-12.5”/, PAGE, and gels were either stained with Coomassie blue R250 (panel A) or blotted to nitrocellulose for probing with rabbit anti-ESgp30 serum (panel B). The antibody-bound markers

protein was visualised

(kDa)

and

lane

DBY747[pYEULCE/30B/7] scanned using a Molecular and to the dimeric

V the

using peroxidase-conjugated

lysate

goat anti-rabbit immunoglobulin and 4-chloro-1-naphthol. Lane M contains the size for 2 h. Other lanes contain lysates of of DBY747[pYEULCE] control cells induced with Cu*’ the Coomassie blue-stained gel was with Cu * + for the times indicated above the lanes (h). For quantitation

ceils induced Dynamics (Sunnyvale,

non-glycosylated

ESgp30

CA) computing

(closed

arrowhead),

densitometer. having

apparent

Arrowheads molecular

point to the non-glycosylated weights

ESgp30 (open arrowhead)

of 15 kDa and 40 kDa, respectively.

111 SDS-PAGE gels. This protein corresponds to the expected 15kDa non-glycosylated primary translation product of the ESgp30 cDNA (Savin et al., 1990). It also forms a tightly bound, approximately 40-kDa homodimer (T.A.A. Dopheide, personal communication) which can be seen as the cellular ESgp30 concentration rises.

REFERENCES Baldari,

C., Murray,

human

interleukin

Davis,

vectors with a high degree of convenience for cloning and for regulated expression of foreign genes. 1. The primary vector, pYEULlacZ, permits cloning into ten unique cloning sites in the pUC19-derived MCS with screening for recombinants in E. coli by monitoring the disruption of IucZ. 2. The pYEULlacZ vector contains both leu2-d and URA3 alleles allowing selection of transformants in both leu2 and ura3 S. cerevisiue strains, thereby lessening the number of constructs required when transforming a variety of different yeast strains. 3. The new plasmids have higher copy numbers than their LEU2-containing progenitor (YEplac 18 1). This may explain the high levels of foreign protein production observed. 4. We have integrated CUP1 B and CUP1 E expression cassettes into pYEULlacZ making new vectors, pYEULCB and pYEULCE, that each have four convenient cloning sites for the Cu2+ -regulated expression of foreign genes. We provide an example of the Cu2 + -regulated production of ESgp30, a secreted glycoprotein from the parasitic nematode Trichostrongylus coluhrformis.

lflin Saccharomyces

R.W., Thomas,

Denhardt,

M., Cameron,

Methods

Enzymol.

C.L.:

of a fragment

cerevisiue. EMBO

J. 6 (1987)

plementary

DNA.

for enzymatic

S. and

and hybridization

65 (1980) 404-41 I.

D.T.: A membrane-filter

technique

Biochem.

for the detection

Biophys.

Res.

Commun.

of com23 (1966)

641-646. Gietz,

R.D. and Sugino,

A.: New yeast-Escherichiu

with in vitro mutagenized

coli shuttle

vectors

yeast genes lacking six-base pair restriction

sites. Gene 74 (1988) 527-534. Heusterspreute,

M., Oberto,

with restriction siue shuttle

Jagadish,

J., Ha-Thi,

site banks,

vectors.

V. and Davidson,

III. Escherichia

P.R., Irving, R.A., Azad, A.A. and Macreadie,

and characterization

virus polyprotein

in yeast. Gene 95 (1990) 179-186.

U.K.: Cleavage

of structural

the head of bacteriophage Macreadie,

LG., Jagadish,

Macreadie,

and copper-inducible

lads.

Rose, A.B. and Broach, in yeast:

of

expres-

Acids Res. I8 (1990) 1078. P.R.: Versa-

expression

of proteins

21 (1989) 147-150. O., Vaughan,

expression

Kluyveromyces

disease

the assembly

for cloning

for the copper inducible

I.G., Horaitis,

Constitutive

during

M.N., Azad, A.A. and Vaughan,

designed

in yeast. Plasmid

proteins

bursal

227 (1970) 680-685.

I.G.: Yeast vectors

tile cassettes

of infectious

T4. Nature

sion of foreign genes. Nucleic Macreadie,

cerevi-

Gene 34 (1985) 363-366.

M.N., Vaughan,

Laemmli,

J.: Vectors

coli-Succhuromyces

I.G.: Expression

P.R. and Clark-Walker,

ofthe Succhuromyces

G.D.:

cerevisiue CUP1 gene in

Yeast 7 (1991) 127-135. J.R.: Propagation

2-pm circle-based

vectors.

and expression Methods

of cloned genes

Enzymol.

(1990) 185,

234-279. Sambrook,

J., Fritsch,

Laboratory

Manual

Press, Cold Spring Savin, K.W., Dopheide,

E.F. and Maniatis, (2nd

Edn).

Harbor, T.A.A.,

Cold

T.: Molecular Spring

activity

Frenkel,

of a 30 kDa glycoprotein

Trichostrongylus

colubriformis.

Cloning.

Harbor

A

Laboratory

NY, 1989. M.J., Wagland,

W.N. and Ward, C.W.: Characterization, ACKNOWLEDGEMENTS

G. and Galeotti,

J., St. John, T.P., Scherer,

R.A.: Rapid DNA isolation

analysis.

new E. cob-S. cerevisiue shuttle

G., Cesareni,

229-234. Padgett,

(e) Conclusions We have constructed

J.A.H., Ghiara,

A novel leader peptide which allows efficient secretion

B.M., Grant,

cloning and host-protective

secreted

by the parasitic

Mol. Biochem.

Parasitol.

stages of 41 (1990)

167-176.

We thank Drs. Daniel Gietz for YEplac plasmids, David Botstein for strain DBY747 and Drs. P. Vaughan and A. Azad for helpful discussions. The assistance of Alister Ward and Kaye Swiney (Department of Biochemistry, Melbourne University) with the computing densitometry is gratefully acknowledged.

Towbin,

H., Staehelin,

proteins

T. and Gordon,

from acrylamide

some applications.

J.: Electrophoretic

gels to nitrocellulose

transfer

sheets: procedure

of and

Proc. Natl. Acad. Sci. USA. 76 (1979) 4350-4354.