Effect of vector type, host strains and transcription terminator on heterologous gene expression in yeast

Vol. 140, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

October 30, 1986

Pages 602-608

EFFECT OF VECTOR TYPE, HOST STRAINS AND TRANSCRIPTION ON HETEROLOGOUS GENE EXPRESSION IN YEAST Kong-Bung

Choo,

Sheue-Mei

Wu, Li

Hung,and

Recombinant DNA Laboratory, Department Veterans General Hospital, Taipei, Taiwan Received

September

10,

TERMINATOR

Hsien-Hsiung

Lee

of Medical Research, 112, Republic of China

1986

Using the surface antigen gene of the hepatitis B virus, and the promoter and terminator sequences of the yeast pho5 gene as a a series of closely related expression plasmids were model system, constructed to investigate the effect of vector type, genetic background of host strains and the presence of transcription terminator on the expression of heterologous,gene in yeast. Plasmids carrying the replication origin of the 2~ plasmids were found to be much more stable than those either independently or simultaneously Gene expression was also higher with 2vmcarrying a&51 sequences. based plasmids. Yeast selection marker (ttlpl or eeu2) and therefore the host strains used did not have significant effects on gene expression. Addition of transcription terminator sequences downstream to the HBsAg gene also contributed only limited increases Q 1986 Academic Press, Inc. in gene expression levels. A wide

range

of biological

gene

expression.

have

several

with

no known pathogenicity

Among the distinct

man in brewery romosomal

hosts

industries.

systems

yeast

transformation

procedures

2) and a variety

of efficient

vectors

have

been

to suit

have

have

specific

described.

are

used

for

this

long

been

purpose,

yeasts

promoters

(3-5)

vectors

most

associated

studied

More

have now been well

Yeast

heterologous

with

and extrach-

been well

purposes.

for

common micro-organisms

The nuclear

of yeasts

manipulated

been

used

They

and they

easily

(1,

being

advantages.

and bakery

genetic

hosts

have

and are

importantly, established

and yeast commonly

cloning used

for

Abbreviations: a)Ldl, autonomously replicating sequences of yeast of replication of the 2pm plasmid; chromosome; 2um ohi, origin HBsAg, hepatitis B surface antigen; (k)b, (kilo)base pairs; Pi, inorgainc phosphate; S/N, ratio of sample value to negative value in radio-immunoassay for HBsAg. 0006-291X/86 Copyrighr All rights

$1.50 0 I986 b-k’ Academic, Press. oj reproduction in any ,fbrm

Inc. reserved.

602

Vol. 140, No. 2, 1986

gene

BIOCHEMICAL

expression

originally

have

cloned

carrying

the

plasmid

In

two types

related

carrying

yeast

constructed

to

of the

to systematically on gene

on gene

test

the

and the

the

sequences

(6)

and vectors

expression,

the

effects

of

of closely

of host

Plasmids

strains

were also

genetic

transcription

2pm

effect

a series

of different of

endogenous

compare

expression.

presence

a,tbl

yeast

to minimize

effect

RESEARCH COMMUNICATIONS

chromosomes

of replication

were designed

variables

MATERIALS

the

order

plasmids

strains

those

of vectors

and other

host

from

origin

(7).

these

been

AND BIOPHYSICAL

background

terminator

of

sequences.

AND METHODS

E,coLilyeast shuttle plasmids were Construction of shuttle plasmids: constructed according to standard procedures(8). E.coLi strain DH5 (9)was used in all E.co.& transformation experiments. The plasmids were transformed into yeast cells using the method of Ito et n!(2). Analysis of yeast transformants: In each experiment, transformants were grown in 10 1 f a mixture of phosphate-depleted YEPD (1% yeast extract, 2%mpeitone, 2% glucose) and minimal glucose medium (0.67% yeast nitrogen base, 2% glucose, 20 mg/ml uracil) in 1:4 ratio in 50 ml conical flasks at 30" for 36-40 h with vigorous rotary shaking at 200 rpm to A600 of 5-10. The cells were homogenized with glass beads and the homogenates were assayedfor HBsAg using the AusriaR kit from Abbott Laboratories as described (10). RESULTS AND DISCUSSION As a model host

strains

promoter were

for

the

investigation

and transcription

sequences

placed

of the

upstream

fragment

sequences

entire

to a 1.4 kb EcoRI-SaLI SalI of

site

to obtain

site

This

clone gene

(p2~-Sll) in

the

promoter

out

was corrected

and digesting

the

SueI

was obtained correct

acid

on gene

reading

in

of vector

(pho5)

two stages

(12)

carrying

the

This

rendered

of phase

using

by linearizing termini which frame(l0).

with

the

Bat31

gene

(Fig. pku5

was first

the upstream 116-5

The BaL31

1).

A

ligated the

reading

frame

pho5

initiation

at the

and religating.

showed expression

(11)

coding

HBsAg gene via

clone

type,

expression,

83 bp of the region

116-5.

effect

phosphatase

containing

fragment

the HBsAg sequence

codon.

yeast

clone

the

terminator

to the HBsAg gene

2.7 kb EcuRI-SaLI and the

of

of digestion

S&I A

the HBsAg resulted

Vol. 140, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E.COIi

-

60131

1

w5

----_

p2p-SI

I

--em--

BE

H l.gKb-

phob terminator

E 8, .

3.OKbd 5SKb -

E

IL---

vql7,PW2-F*

psy97

YRp7-SllT p~u2-SI IT pShl97-Sl

Fig.

in retention gene,

IT

1. Construction of yeast expression plasmids for HBsAg gene. Clone 116-5 was first constructed by assembling the various components the origin of which is described in Fig. 2.and in the text. The excess sequences between the pho5 promoter and the HBsAg gene were removed by linearizing 116-5 with SaLI and digestion with B&Z31 to obtain p21+1-S11. The 1.9 kb BumHI fragment from p2u-Sll was inserted into pLeu2-F (Fig. 2) to obtain pLeu2-Sll. A 1.1 kb HpaII fragment was excised from the 3' region of the pho5 gene, the termini filled in with DNA polymerase and was then inserted at the HpaI(H) site at the 3' -end of the HBsAg gene of p2~-Sll to produce p2~-SllT. The3.0 kb BamHI or the 5.5 kb EcoRI(E) fragment was inserted into YRp7, pLeu2-F and pSM97 (Fig. 2) to obtain YRp7-SllT, pLeu2-SllT and pSM97-SllT. Open box, pho5 coding region; hatched box, HBsAg gene; filled box, pho5 terminator sequences.

of

the

13 amino acid

(13)

and the

entire

K.B.

Choo, unpublished

first

three

residues

amino acid

from the PreS2 region

HBsAg gene in plasmid

2pm o&i from pJDB219(14)

data).

residues

Plasmid

and the tap1 604

p2~-Sll

p2~-Sll marker

also (Fig.

from the

pho5

of HBV genome (H.H.

Lee and

contains 2).

the

Vol.

140,

No

BIOCHEMICAL

2, 1986

AND

(a)

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

(b)

(a) p'Zp-Sll; (b) pLeu2-F; 2. Plasmids used in the present study. source of the various fragments: (A) Cc) pSM97. Original 2.7 kb EcoRI-SaLI fragment from pAP20 (11, 12) containing the pho5 promoter. (B) 1.4 kb SaLI-EcoRI fragment from pTWS105 containing the HBsAg gene (unpublished); (C) 2.5 kb EcoRI fragment from pJDB219 (12) containing the 2urn origin of replication; (D) 4.4 kb icoR1 fragment from YRp7 (13) carrying the thpl and Tc genesbut without the ahnl sequence; (E) 3.3 kb Hind111 fragment from pJDB219 containing the eeu2 gene and the 2pm ori; (F) 2.7 kb Hind111 fragment of pUC9; (G) 1.4 kb EcoRI fragment from YRp7 with thpl gene and atrnl sequence; (H) 5.6 kb EcoRI fragment from SW9 (P. Nagley, personal con-munication) containing the Tcii gene and the ?vm

Fig.

Ohi.

In order terminator

to

the effect

on the expression

sequence

of

was inserted

SllT

the

at

was contained the

the

to obtain

into

F (2um ohi,

2) and pSM97 (both

2).

SllT

The resulting

and pSM-SllT

fragment

which

from p2u-Sll

does

of all columns

These plasmids strains Marked

which

contain

the

in Table

atlbl

pLeu2-F

plasmids

1).

Subfrom p2~-

terminator) pLeu2-

itrpl)(l5),

and 2urn

04i,

YRp7-SllT, the

(Fig.

pLeu2-

promoter-HBsAg was excised

1).

described

thpl)

The major

are summarized

1.

were transformed

were auxotrophic

differences

(a&Al,

the pho5 terminator into

six

downstream

(Fig.

For comparison,

and was inserted

characteristics in left-hand

not

a 1.1 kb

located

were designated

respectively.

terminator

gene-(pho5

vectors'YRp7

plasmids

transcription

3.0 kb EcoRI fragment

sequences was inserted

(Fig.

the

p2~-SllT

(pho5 promoter)-HBsAg

eeu2)(Fig.

of

within

site

HpcrI

1.9 kb BumHI or the

containing

the presence

of the HBsAg gene,

to the HBsAg gene in p2~-Sll sequently,

of

the phu5 gene which

fragment

HpaII

test

into

at the

in the genetic

-0~~1

stability 605

three

different

and/or of

the

host Leu2 loci.

the plasmids

were

t

pLeu2-SllT

t

t

t t

t

t 38

0

96

t

t

100

t

94

52

0

a5

66

a7

70

DB746

n.d.

n.d.

n.d.

n.d.

n.d.c

96

10.9

0

25

16.5

26

20

GN3C. 2

n.d.

n.d.

0 13.4

n.d.

n.d. 47.5

7.4

n.d.

35

20 16.9

YNN27

(S/N)b

DB746

HBsAg expression

of HBsAg gene in yeast

YNN27

Stabilitya

Plasmid GN3C.2

levels

and expression

t

t

Terminator

stability

93

Leu2

marker

plasmid

t

Thpl

Yeast

affecting

thpl

uha31

bTransformants were grown in 10 ml and homogenized with glass beads Abbott Labs. Values are averages c.1+,, and 11-11indicate presence or

[a,

Cell were of low Pi medium (10 mg/l Pi) to A660 = 5 - 10. (450 pm) and the lysates were.assayed for HBsAg using Ausria of 2-3 determinations. absence of the indicated sequences. n.d., not determined.

harvested kit from

aGenetic stability was determined by plating equal aliquots of transformant cells on minimal and rich colonies on YEPD plate) x 100. (Values on minimal plate/no. (YEPD) media. Stability = (No. colonies GN3C.2: [a, ecu2 t&pl]i DB746; [a, hid1 ecu2 thpl uhfd]; YNN27: are averages of 2-4 determinations).

pSM-SUT

t

t

pLeu2-Sll

YRp7-SllT

t

p2~-SllT

ahA

2vm

tc

type

Vector

1. Factors

p2u-Sll

Plasmid

Table

Vol.

140,

No. 2, 1986

observed very

in

BIOCHEMICAL

that

unstable

plasmid

while

some differences

of

the

is

also

reflected

less

expression but

in

DB746,

amounts

plasmids

Our results

yeast

vector

than

ahcll

There

with

used.

probably and are

of

recombination

a4bl

vectors,

results

in

studies

on the loci

(CEN)

reveal used

loss

have

been

and the

sequences HBsAg gene. may still

effect

presence

case,

the

putative

was positioned Localization have

greater

use of 2pm vectors,

important

factor

which

of the

in

250 bp from the

polyA

influence the affects

the

choice

the

signal

expression 607

of stable

In

are the

case

easily Recent

by centromeric did

of the

not

host

on gene

strains expression.

pho5 terminator

closer

expression.

of promoter

type

more

plasmids.

termination

on gene

effect

sequences

terminator of

differ-

2pm plasmids

background

signal

to produce

the

Our results

of transcription

of

is

sequences

17).

of genetic

polyA

that

the

exception

marker.

copy number.

au

(16,

the

significant

chromosomal

sequences

2pm 2-fold

significantly

fact

in high

described

the

hand,

no consistent

expression

are

with

of gene

other

seemed

the most

vectors

stabilization

significant

In our

the

the

for

as selection

gene

present

produced

With

however,

due to the

extrachromosomal

YRp7-SllT

On the

terminator

showed that

2pm-based

vectors

the

or 1~2

and therefore

stability

pSM-SllT

strains.

were,

thpl

therefore

stability

with

the presence

For any plasmid,

different

plasmids

of HBsAg.

between

on plasmid

with

in

HBsAg gene.

higher

with

strains.

The plasmid

is relatively

was

stable

host

average.

do exist.

sequence

reduced

of the

on the

COMMUNICATIONS

more

or no HBsAg while

some variations

of p2~-SllT

ences

expression

HBsAg gene

are observed

utrnl

different

was greatly

plasmids

differences

higher

among the

little

other

of the

plasmids

the

produced

HBsAg than

the

as shown by pSM-SllT.

in

transformants

RESEARCH

carrying

2urn plasmid

sequences

BIOPHYSICAL

were relatively

stability

of the

am1

YRp7-SllT

2pm plasmids

in

The stability

AND

codon to the Thus,

may be the

of foreign

of

genes

the

gene besides

more in

yeast.

Vol. 140, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENT

from

This work was supported by research the National Science Council of the

grant NSC75-0609-B075-01 Republic of China.

REFERENCES 1.

2. 3. 4. 5. 6. 7. a. 9. 10.

11. 12. 13.

:;: 16. 17.

Hinnen, A., Hicks, J-B., Fink, G.R. (1978) Proc. Natl. Acad. Sci. USA 75 1929-1938. Ito, H., Ekuda, Y., Murata, K., Kimura, A. (1983) 5. Bacterial. 153, 163-168. Miyanohara, A., Toh-e, A., Nozaki, C., Hamada F., Ohtoms, N., Matsubara, K. (1983) Proc. Natl. Sci. USA 80, l-5. Hitzeman, R.A., Chan, C. Y., Haige, F.K. et UL. (1983) Nucl Acids Res. I.& 2745-2763. Bitter, G., Chen, K.K., Banks, A.R., Lai, P.H. (1984) Proc. Nacl. Acad. Sci. USA 81, 5330-5334. D., Sherer, S., Davis, R. (1979) Proc. Struhl, K., Stinchcom6; Natl. Acad. Sci. USA 76, 1035-1039. Hollenberg, C.P. (1982) Curr. Topics Microbial. Immunol. 96, 119-114. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor. Hahanan, D. (1985) In: "DNA Cloning: A Practical Approach" (D.M. Glover, Ed.) IRL Press, Oxford, pp109-136. Choo, K.B., Wu S.M., Lee, H.H., Lo, S.J. (1985) Biochem. Biophys Res. Commun. 131, 160-166. (1980) Proc. Natl. Acad. Sci. USA -77 Kramer, R.A., Andersen, N. 6541-6545. Arima, K., Oshima, T., Kubota, I., Nakamura, N., Mizunaga, T., Toh-e, A. (1983) Nucl. Acids Res. ll-, 1657-1672. 2. lichel, M-L., Sorigo, P., Tiollais, P., Dejean, A., Brechot, C., Michel, Wain-Hobson, S. (1984) In: "Viral Hepatitis and Liver Diseases" (G.N. Vyas, J.L. Dienstag, J.H. Hoofnagle, fnagle, Eds) Grunt Fr Stratton, Inc., N&w York, ~~49-66.~ Beggs J.D. (1978) Nature 275, 104-109. Stinchcomb, D.T., Struhl, K., Davis, R.W. (1979) Nature E, 39-43, Dani, G-M., Zakian, V.A. (1983) Proc. Natl. Acad. Sci. USA Q 3406-3410. Larionov, V.,Kouprina, N., Karpova, T. (1984) Gene 28, 229-235.

608