Gene, 101 (1991) 105-111 0

1991 Elsevier

GENE

Science

Publishers

B.V. 0378-I 119/91/$03.50

105

04028

Mutations of the a-galactosidase by yeast DNA;

(Recombinant

site-directed

signal peptide which greatly enhance secretion of heterologous proteins

Saccharomyces

mutagenesis;

cerevisiae;

expression

vector;

regulated

expression)

Kathryn J. Hofmann and Loren D. Schultz Department of Cellular and Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486 (U.S.A.) Received by J.A. Gorman: 24 July 1990 Revised: 15 November 1990 Accepted: 25 December 1990

SUMMARY

The Saccharomyces carlsbergensis MEL1 gene encodes a-galactosidase (melibiase; MELl) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MELI promoter and MEL1 sp coding sequence (MELIsp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MELlsp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MELIsp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI- to levels similar to those achieved when using the yeast MFal pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase

in the secretion

levels of those two proteins.

INTRODUCTION

The S. carlsbergensis MEL1 gene, which encodes a-galactosidase (MELl), has several properties which make it

Correspondence to: K.J. Hofmann, Biology,

Merck

Sharp

PA 19486 (U.S.A.) Abbreviations: complete

aa, amino

Echis, echistatin; gene encoding

acid(s);

lacking EndoH,

MELI;

MFclZ, gene encoding

MELl,

CmR,

a-mating

dodecyl unit(s);

sulfate;

sp, signal

wt. wild type.

synthetic or 1000 bp;

(melibiase);

factor; mu, milliunit(s); PAI-1, plasminogen

fluoride; peptide(

MELZ,

coding sequence; nt, nucleotide(s);

ori, origin of DNA replication;

electrophoresis;

phenylmethylsulfonyl

CM-leu,

H; kb, kilobase

cc-galactosidase

tor type 1; PolIk, Klenow (large) fragment PMSF,

West Point,

chloramphenicol-resistant;

MEL1 signal peptide

oligo, oligodeoxyribonucleotide; polyacrylamide-gel

bp, base pair(s);

leucine;

MELIsp,

Laboratories,

Fax (215)661-7320.

endoglycosidase

antibody;

of Cellular and Molecular

Research

Tel. (215)661-6611;

medium

mAb, monoclonal

Department

and Dohme

activator

PAGE, inhibi-

of E. coli DNA polymerase

S., Saccharomyces; TCA,

I;

SDS, sodium

trichloroacetic

acid;

U,

attractive for the development of a yeast secretion system. The MEL1 promoter is induced by galactose or melibiose and repressed by glucose in a fashion similar to the genes (e.g., GALI, GALIO) involved in galactose metabolism (Post-Beittenmiller et al., 1984). In addition, MEL1 is a secreted protein which can account for up to 40% of the total protein secreted into the medium (Sumner-Smith et al., 1985). As expected for a secreted protein, MEL1 has an 18-aa sp which shares common features with other characterized eukaryotic sp (Liljestrom, 1985) and relies exclusively on signal peptidase for cleavage. Since the MEL1 sp has been used successfully to secrete a Cellulomonasfimi exoglucanase from S. cerevisiae (Curry et al., 1988), it was of interest to determine whether the MEL1 sp would direct the efficient secretion of two very dissimilar proteins: Echis and human PAI-1. Echis, a potent inhibitor of ADP-stimulated platelet aggregation, is a 49-aa polypeptide containing eight cysteines and four disullide bonds which are critical for biological activity

106

(Gan et al., 1988). of both tissue-type (Alessi et al., 1988) no cysteine residues

Human PAI-1, a glycoprotein inhibitor plasminogen activator and urokinase is a 379-aa polypeptide which contains and has three potential N-linked glyco-

sylation sites (Ny et al., 1986). In this report, we show that the wt MEL1 sp is very inefficient for directing the secretion of either Echis or PAI- 1. On the other hand, we report the construction of two mutant MEL1 sp which efficiently direct the secretion of both Echis and PAI-1.

RESULTS

AND DISCUSSION

(a) Construction of expression vector with MELIsp To evaluate the utility of the MEL1 sp for directing the secretion of heterologous proteins from yeast, the expression vector pKH6-2A was constructed (Fig. 1). The expres-

I

amHl

III

Pst1

sion cassette

in this vector contains

(1) the galactose-regu-

lated MEL1 promoter fused to the MELlsp; (2) a unique Sac1 cloning site which allows the precise fusion of the coding sequence for a desired protein to the MELlsp, and (3) MFcd transcriptional termination sequences. Furthermore, this vector contains the bacteriophage fl intergenic region and ori which facilitate preparation of singlestranded DNA for site-directed mutagenesis (Vernet et al., 1987). (h) Expression of echistatin in yeast Since previous studies had shown that the yeast MFal pre-pro leader can direct the secretion of Echis from yeast (Jacobson et al., 1989) we decided to evaluate the MEL1 sp for secretion of Echis. The vector pKH6-2A/Echis (Fig. 1) was constructed with the coding sequence for aa l-49 of mature Echis (Glu’ ‘) precisely fused to the MELlsp (Gly + ‘). For comparison, the vector pKH4-3B/Echis was constructed which is similar to pKH6-ZA/Echis but uses the MFc(1 pre-pro leader to direct secretion (Carty et al., 1990). Transformants of strains BJ1995 and JRY188 were grown in liquid cultures and induced with galactose (Fig. 3 legend). As seen from analysis of media supernatants

Pst I

at the ,SphI site within the yeast 2~ sequence.

the filled-in BarnHI

Sac I

Pst I

within the plasmid

T4 DNA polymerase

BamHI

SfiI

Bgl II

as a 0.56-kb the

terminator pKH6-fl

1

PstI

cassette

fragment

yeast 2~ and pBR322

of pKH6

to form pKH6-fl.

Pstl-BarnHI pTZ-Echis encoding

The original

into the nt sequence

fragment

fragment Pst I

encoding

(Jacobson

encoding

containing

site of pKH6-2A

fragment

Echis (Glu’).

were inserted

containing

the

The 1.2-kb SjiI-BglII (Garde11

to yield a 1.25-kb blunt for mature

PAI-I

into the SacI(filled-in)-BglII

to yield the expression These

from

with an oligo adapter

the entire coding sequence

respectively.

cleavage

The 0.13-kb

of Echis was isolated

with an oligo adapter

(Val’). These two fragments pKH6-2A/PAI,

site within

a unique Sac1

all but the first 10 aa of mature human PAI-

et al., 1990) was ligated Bg/II fragment

Sac1

vector pKH6-2A.

aa IO-49

for mature

promoter-

at the signal peptidase

et al., 1989) and ligated

sequence

sequences

the

mutagenesis,

aa l-9 to yield a 0.15-kb blunt BarnHI

entire coding KHG-PAfEchis

containing

was deleted and using site-directed

site ofthe MEL1 sp to yield the expression

1

Pst I

and ligated with the 7.5-kb PstI-SphI

of pKH6 containing

site was introduced

T4 DNA ligase

T4 DNA ligase

blunt PstI fragment

3.9-kb

Inc.) and the EcoRI site

was filled-in with PolIk. The fl on’ was then isolated

(filled-in) fragment and

PstI linkers were ligated to

site of pUCf1 (Pharmacia,

vectors

two

gene

pKH6-2A/Echis fragments

were

and also

Fig. 1. Construction and structure of pKH6-2A-related expression vectors. The plasmids pKH6-2A, pKH6-ZA/Echis and pKH6-2A/PAI were

inserted into the SphI(filled-in)-BglII site ofpKH4-3B (Carty et al., 1990) to yield the expression vectors pKH4-3B/Echis and pKH4-3B/PAI (not

constructed

shown) which are similar to the pKH6-2A

MEL1

as follows.

promoter

(Sumner-Smith ing the remaining

A 0.54-kb

and the first

EcoRI-SphI

fragment

10 aa of the MEL1

et al., 1985), a 37-bp SphI-BamHI

encoding

the

sp from pMP550

oligo adapter

encod-

8 aa of the MEL1 sp and a 0.40-kb BarnHI-Hind111

EcoRI-Hind111

fragment.

The 1 l.O-kb BumHI-Sal1

fragment

of

the vector pCl/l (Rosenberg et al., 1984) and the 0.98-kb EcoRI-Hind111 fragment described above were made blunt-ended with PolIk and ligated to form pKH6. To facilitate of pKH6 was constructed

site-directed containing

mutagenesis,

restriction Pharmacia,

fragment encoding a three-frame translational terminator plus the MFal transcriptional terminator (Schultz et al., 1987) were ligated to form a 0.9%kb

GAL10 promoter

and coding sequence

endonucleases

and

versions

but contain

the yeast

for the MFal

pre-pro

leader. All

DNA-modifying

Inc., or New England

Biolabs,

enzymes

(vertical stripes), MELlsp (blackened box), three-frame terminator (open box), MFctJ transcriptional terminator stripes), fl ori(jagged

were

from

Inc. Key: MEL1 promoter translational (horizontal

line), pBR322 (thin line), yeast leu2-d gene (shaded

box), yeast 2p sequences (chain (ECS), direction of transcription

a modified version

represent

Echis or PAI-

the phage fl ori effectively inserted

represent

deleted

cloning

of small circles), synthetic Echis gene (arrow). The heavily stippled boxes

coding sequences sites.

as indicated.

The parentheses

107 TABLE

(c) Construction

I

Effects of sp mutations

on the secretion

Plasmid a

of Echis

Echis secreted

(rig/A,,,)

b

BJ1995 0

0

173

150

pKH6-2A pKH4-3B/Echis

JRY188

10

19

pKH6-2A/Echis-ml

273

229

pKH6-2A/Echis-m2

6

6

pKH6-2A/Echis-m3

202

131

pKH6-ZA/Echis

3 Transformants

of BJ1995 and JRY188

mids were grown, harvested, as described b Aliquots

in Fig. 3 legend (for plasmids of concentrated

culture

etal.,

1.2A,,,

corresponding

to the vol-

units of cells were assayed

1989). The Echis concentrations

were divided

by the A.&ml

plas-

were concentrated

binding of [ ‘a51]Echis to activated

Echis by competitive

(Jacobson

the indicated

see Figs. 1 and 2).

media supernatant

ume of original culture containing secreted

containing

and media supernatants

for

platelets

in ng/ml of original

of the culture.

(Fig. 3; Table I), there was negligible secretion of Echis directed by the wt MEL1 sp. In contrast, the MFcr 1 leader directed the secretion of 150-170 ng Echis/A,,, unit of cells, depending on the strain used (Fig. 3; Table I). This corresponds to 0.4-0.8 pg Echis/ml (results not shown).

-18

wt

fll

-17

-16

-15

-14

-13

-4

-3 -2

-1

Ala

Phe

Tyr

Phe

-12 -11 -10 -9 -8 -7 -6 Thr Ala Cys Ile Ser Leu

-5

Met Phe

Leu

Lys

Gly

Val

Phe

Gly

ATG

TTT

GCT

TTC

TAC

TTT

CTC

ACC

GCA

TGC

ATC

AAG

GGC

GTT

TTT

GGG

Met

A?J

Ala

Phe

Leu

Phe

Leu

Thr

Ala

Cys

Ile Ser Leu Lys Gly Val Phe

Gly

Phe

Leu

Thr

Ala Cys Ile SW Leu pro Gly Val Phe

Gly

AGA

m2

Met

AGT

TTG

TTG

Phe

Ala

Phe

Tyr

CCA

m3

Met

ti

Ala

Phe

AGA

Fig. 2. Nucleotide mutant

Leu

Phe

Leu

Thr

Ala

Cys

Ile Ser Leu pro Gly Val Phe

TTG

sequence

and deduced

MEL1 sp. For mutagenesis,

relA; pCJlO5

(CmR)]

pKH6-2A/Echis

aa sequence

The M13K07

was used according

to the manufacturer’s

phage, preparation

of single-stranded (Muta-Gene@

with the pKH6-2A/PAI

and

helper phage (Pharmacia,

Inc.)

Precipitation

of the

DNA and the mutagenesis

directions.

proce-

in vitro Mutagenesis

Inc.) except that two oligos were used as primers reaction

at a 25 : 25 : 1 primer

for the wt and

the E. coli strain CJ236 [dut, ung, thi,

was transformed

plasmids.

dure were as described

Gly

CCA

Kit, Bio-Rad,

in a single mutagenesis

: primer : template ratio. The oligo primers

used were: oligo 1: 5’-CGGTGAGAAACAAGAAAGCTCTCATCG5’-CCAAAAACGCCmCAoligo 2: TCGTTG-3’; and AACTGATGC-3’. underlined.

The mismatches

Mutations

sequencing

method

mids bearing

were

verified

with the wt MELZ sequence

DNA

(U.S. Biochemical

Plas-

using Sequenasecn

the mutations

derived from the indicated

nated by the following

suffixes: oligo 1, -ml;

2, -m3. For simplicity,

only the mRNA-like

codons

are shown and the aa substitutions

above the wt sequence cleavage

site.

are

by the chain-termination

show the location

Corp.).

oligos are desig-

oligo 2, -m2; oligos 1 plus nt strand

and the mutated

are underlined.

The numbers

relative to the signal peptidase

of mutants of MEL1 sp

A comparison of the aa sequence of the MEL1 sp (Fig. 2) with the typical structure of eukaryotic sp (von Heijne, 1985) leads to the following observations. First, the MEL1 sp has a positively charged aa (Lys) in the C-terminal (c) domain instead of the more typical presence of a positively charged aa in the N-terminal (n) domain. Second, a Lys residue at position -5 replaces a more typical Pro residue at this position. Third, the hydrophobic core (h) domain is frequently disrupted by nonhydrophobic residues resulting in stretches of only one to two hydrophobic residues throughout most of the core. On the other hand, the MEL1 sp Gly - ’ and Val 3 have a good Iit with the -1, -3 rule proposed by von Heijne (1984). To address these considerations, three modified versions of the MELlsp were prepared in the pKH6-2A/Echis vector by site-directed mutagenesis (Fig. 2 legend). The deduced aa sequence for the mutant sp is shown in Fig. 2. Mutant ml has Phe ” -+ Arg and Tyr - I4 + Leu changes, resulting in an sp with a positive charge in the n-domain and a more hydrophobic h-domain than the wt MEL1 sp. Mutant m2 has a Lys - 5 --+ Pro change, resulting in the removal of a positive charge and replacement with a helix-breaking Pro residue which is more typically found at position -5 (von Heijne, 1985). Mutant m3 is a combination of mutants ml and m2. (d) Echistatin secretion directed by mutants of the MEL1 sp Strains BJ1995 and JRY188 were transformed with the three mutant derivatives of pKH6-2A/Echis, and transformants were evaluated for the secretion of Echis. A heavily stained 5-kDa band was seen on gels of media samples from cultures of cells containing the expression vectors with mutations ml or m3 (Fig. 3). This band comigrates with purified, synthetic Echis and appears to be properly processed Echis. The ml sp directs secretion of 2200 ng Echis/A,,, unit of cells in both strains as judged by comparison with the silver-staining intensity of pure Echis (Fig. 3) and the bioassay results (Table I). The single mutant ml appears more effective than the double mutant m3; this difference seems more pronounced in JRY 188 than BJ 1995. The expression vectors containing the wt or m2 sp coding sequences direct secretion of negligible amounts of Echis only. The qualitative similarity between the bioassay and silver-stain results suggests that most of the secreted Echis is biologically active. Since correct folding and disulfide-bond formation are essential for biological activity (Gan et al., 1988) these data suggest that the mutant MEL1 sp direct the secretion of Echis in its native, correctly folded form. The data also indicate that the ml sp functions as well as the MFcrl leader in directing the secretion of Echis (Table I).

108

kDa

--.

46 -

r;

30. 21 14 * 6.5 *-

**I*

1 Fig. 3. Secretion of California,

3

4

5

6

12

3

4

5

6

1

of Echis by yeast. The S. cerevisiae strains JRY188 (MAT&, sir3-8,leu2-112, trpl, ura3-52, hid, cir’),

Berkeley,

cir” by standard

2

and BJ1995 (MATa, leu2, trpl, ura3, prbl-1122, pep43,

techniques.

Yeast transformants

were propagated

ofA 6,,O= 1.3-2.3, the cells were harvested were harvested by centrifugation. Media

supernatants

resuspended

ml) of distilled water.

in a minimal volume (0.1-0.2

&-

w

3.4 *

by centrifugation

cells were boiled in sample buffer and electrophoresed

in CM-leu

(Schultz

and resuspended

were desalted

et al., 1987) containing containing

columns

corresponding

conditions

3

2% (w/v)glucose

2% (w/v) galactose.

(Pharmacia,

Inc.),

PAGE

University,

were made

at 30°C. At a cell density

After 24 h at 30°C

concentrated

to the volume of original culture

in 0.1 y0 SDS-20%

4

a gift from Dr. J. Rine, University

a gift from Dr. E. Jones, Carnegie-Mellon

in CM-leu

on NAP-25

Samples

under reducing

cir’),

2

the cells

by lyophilization

containing

0.12 A,,,

gels using the PhastSystem

and

units of

(Pharmacia,

in the PhastSystem Development Unit (Pharmacia, Inc.) as described by Heukeshoven and Dernick (1988). Media supernatants were from BJ1995 (panel A) or JRY188 (panel B) containing pKH6-2A (negative control; lane 6) or the pKH6-2A/Echis_relatedvectors with the indicated sp: wt MEL1 (lane 2), m3 (lane 3), ml (lane 4) or m2 (lane 5). Lane 1 (panels A, B and C) contains 25 ng purified, synthetic Echis (kindly Inc.). The gels were silver-stained

provided transformed

by Dr. V. Garsky,

Merck

with pKH4-3B/Echis

Sharp (MFal

and Dohme

Research

Labs.).

For comparative

leader) is shown in lane 4. Protein

(e) Secretion of human PAISince the mutants ml and m3 sp efficiently direct the secretion of a small, unglycosylated protein such as Echis, it was of interest to determine whether they also would direct the secretion of a larger, glycosylated protein such as human PAI-1, a 42-kDa polypeptide with three potential N-linked glycosylation sites and many potential sites for O-linked glycosylation. The expression vector pKH6-2A/ PA1 contains the coding region of mature PAIfused in-frame with the wt MELlsp (Fig. 1). This plasmid was modified by site-directed mutagenesis to yield three derivative plasmids containing the MELIsp mutants described above (Fig. 2). For comparison, the vector pKH4-3B/PAI was constructed (data not shown) which uses the MFcll pre-pro leader to direct secretion. Transformants of strains JRY 188 and BJ 1995 were evaluated for secretion of PAI- 1. Transformants containing pKH6-2A/PAI with the wt MEL1 or m2 sp secreted no detectable PAI- ; however, transformants containing pKH6-2A/PAI with either the ml or m3 sp secreted considerable amounts of PAI- crossreactive protein (Fig. 4) with strain BJ1995 secreting more PAI- than does JRY188. In contrast to the results seen for secretion of Echis, the m3 sp appears to be severalfold more effective than the ml sp for directing secretion of PAI(Fig. 4).

size markers

purposes,

a sample

(kDa) are shown

from the culture

of BJ1995 (panel C)

on the left margin.

(f) Secreted PAI- is hyperglycosylated Transformants containing plasmids with either the ml or m3 sp secrete several discrete PAI- species (48-55 kDa) and many larger PAIspecies (70-150 kDa). The latter appear to represent hyperglycosylated forms of PAI- since this high-M, material disappears and the total amount of smaller (< 50 kDa) PAIspecies increases following EndoH treatment (compare Fig. 4 and Fig. 5). Transformants of JRY 188 and BJ 1995 containing the MFcr 1 leader plasmid, pKH4-3B/PAI, also secrete several discrete PAIspecies (48-55 kDa) and a considerable amount of very large (> 100 kDa) heterogeneous species that also appear to represent hyperglycosylated forms of PAIbased on EndoH digestion results (Fig. 5). The 48-55-kDa PAIspecies presumably contain only core oligosaccharides at the potential N-linked sites in PAI-1. Interestingly, both the discrete 48-55-kDa species and the large (> 70-kDa) forms of secreted PAI- show biological activity as measured by reverse fibrin autography (Erickson et al., 1984; data not shown). Following EndoH treatment of the PAIsecreted by BJ 1995 cells containing the mutant ml or m3 sp plasmids, there is a major band at 43 kDa and a lighter, diffuse band at 45-48 kDa. The major band at 43 kDa appears to represent PAI- that has been processed to remove the MEL1

109

kDa 97 69

69

*

46 PAI-

1234567 Fig. 4. Secretion harvested trated

of human

as described equivalent

PAI-I by yeast. Cell cultures

through

Centricon

to the volume

were concen-

10 filters (Amicon,

of original

culture

Inc.), and

containing

6 A,aO

units of cells or 2 A 6o,, units ofcells (panel B, lane 2) were boiled in sample buffer and electrophoresed ing conditions. (Towbin

Proteins

in 0.1 y0 SDS-9.0% were

et al., 1979) which

PAGE gels under reduc-

electroblotted was then

to a nitrocellulose

blocked

filter

for 1.5 h at 22°C

1 x TTBS (0.02 M Tris . HCI pH 7.6/0.5 M NaCl/O.l y0 Tween-20) 5%

nonfat

dry

milk.

1 x TTBS, 2% nonfat mAb

(American

Following

Diagnostica,

washed

and incubated

England

Nuclear,

overnight

dry milk containing with

‘?-labeled

Inc.). Results

are shown

(panel A) or BJ1995 (panel B) transformed pKH6-2A

(negative control;

with the indicated

22°C

in

No. 379), the filter was

goat

anti-mouse

IgG

for cell cultures with pKH4-3B/PAI

(New

of JRY188 (lane 2),

lane 3) or the pKH6-ZA/PAI-related

25 ng of purified, nonglycosylated

by Dr. S. Gardell,

Labs.) which had been expressed 1990). Protein

at

vectors

sp: wt MELZ (lane 4), ml (lane 5), m2 (lane 6), m3

(lane 7). Lane 1 contains (kindly provided

incubation

in plus

10 ng/ml of a PAI-l-specific

Inc., product

size markers

Merck

Fig. 5. EndoH

analysis

of secreted

PAI-1. Cell cultures

harvested

(Fig. 3 legend) and media supernatants

described

in Fig. 4 legend. Ahquots

equivalent

to the volume of original culture containing

were

digested

Mannheim

with

digestion,

80 pg bovine were

Na . deoxycholate, Samples with

serum

precipitated

are concentrated

pKH4-3B/PAI

was added

0.1 vol.

of

100%

and immunoblotted

supernatants

as a carrier, TCA,

the

4 mg/ml

(lane 3), or the

pKH6-2A/

sp:wt MELZ (lane 4), ml (lane S),

m2 (lane 6), m3 (lane 7). Lane 1 contains PAI-1. Size markers

as in Fig. 4. The

from BJ1995 cells transformed

(lane 2), pKH6-2A

vectors with the indicated

sylated human

(Boehringer-

and the protein pellet was washed twice with acetone.

were electrophoresed

PAI-related

4 A,,, units of cells

pH 5 for 18 h at 37°C. After

albumin

with

as

media supernatant

63 ng) of EndoH

Corp.) in 60 mM Na. acetate

proteins

samples

of concentrated

2.5 mU (approx.

were grown and

were concentrated

25 ng of purified,

nonglyco-

(kDa) are shown on the left margin.

human PAI- 1

Sharp and Dohme

intracellularly

1234567

were grown and

in Fig. 3 legend. Media supernatants

by ultrafiltration

samples

30

1234567

Research

in yeast (Garde11 et al.,

(kDa) are shown on the left margin.

sp. The slightly slower mobility of the EndoH-treated PAI- 1 compared to the unglycosylated, internally expressed PAIcontrol (Fig. 5, lane 1) is probably due to the presence of several sugar residues that remain attached to the polypeptide since EndoH cleavage leaves a single N-acetylglucosamine residue on each site where an oligosaccharide

had been present. The small amount of heterogeneous material at 45-48 kDa probably reflects O-linked glycosylation of Ser and Thr residues in PAI- 1. Thus, the ml and m3 sp appear to direct the secretion of a significant amount of processed PAI-1. Similar results for the various constructions were seen in strain JRY188 (data not shown). Following EndoH treatment, the PAIsecreted by BJ1995 containing the MFcrl leader plasmid pKH4-3B/ PA1 resolved into a major species (56 kDa) with minor amounts of a 45-kDa species (Fig. 5). The 56-kDa species

110

appears to represent unprocessed PAI-I containing the MFG~I leader due to inefficient processing by the KEX2 protease. Since similar results have been seen for host strain JRY 188 (data not shown), this inefficient processing likely is a function of the structure of the fusion protein rather than the host strain used. (g) Analysis of intracellular PAI-

Analysis of intracellularly accumulated forms of PAIcould help to identify the rate-limiting step(s) for secretion of this protein. Thus, to determine whether the mutations in the MEL1 sp affect the intracellular accumulation of PAI- precursors, cell lysates were prepared from BJ 1995

kDa

(h) Conclusions

97 * 69 *

1234567

1234567 Fig. 6. Immunoblot

analysis

grown and harvested resuspended acid-washed

Protein

4 mM PMSF

to remove

Assay)

immunoblotting transformants pKH4-3B/PAI

PAI-1.

Cell cultures

(0.1 M Na phosphate

and broken

glass beads (0.4-0.5

and the samples

centrifuged

of intracellular

(Fig. 3 legend). Cell pellets (20-25 A,,,

in 250 ~1 PC buffer

NaCl) containing to 0.5:/,

and JRY 188 cells containing the various plasmids. Immunoblot analysis shows that a single, approx. 44-kDa species accumulates in cells containing pKH6-2A/PAI with the wt MEL1 or m2 sp (Fig. 6). A doublet band consisting of the 44-kDa species and a slightly smaller one accumulates in cells containing the m3 sp plasmid and in BJ 1995 cells with the ml sp plasmid. The 44-kDa species probably represents unprocessed PAI- (sp still attached) while the smaller species is processed PAI-1. Although coreglycosylated forms of PAI- appear to be secreted into the culture medium {Fig. 4), no detectable core-glycosylated PAI- appears to accumulate intracellularly (Fig. 6). This suggests that the rate-limiting step(s) for secretion of PAI- 1 appear(s) to be prior to the addition of the core-oligosaccharide. This result plus the observation that the straiq’plasmid combinations that secrete little or no PAIcontain only the largest intracellular species suggests there may be very inefficient translocation of these fusion proteins into the endoplasmic reticulum. Therefore, the mutant ml and m3 sp may either improve the efficiency of translocation into the endoplasmic reticulum or, alternatively, may be processed more e~ciently by the yeast signal peptidase following translocation.

mm diameter).

were vortexed

cell debris

were resolved

by vortexing

by 0.1%

as in Fig. 4. Results

pH 7.2/0.5 M for 2 min with

Triton X- 100 was added

for 1 min. The samples

and aliquots

were

units) were

(50 fig protein,

SDS-9.0% are shown

PAGE

were

Bio-Rad

followed

for cell lysates

by

from

of JRY188 (panel A) or BJ1995 (panel B) containing (lane 2), pKH6-2A (lane 3) or the pKH6-2A/PAI-related

vectors with the indicated

sp: wt MEL1 (lane 4), ml (lane 5), m2 (lane 6),

m3 (lane 7). Lane 1 contains PAI-1. Size markers

25 ng of purified,

nonglycosylated

(kDa) are shown on the left margin.

human

(1) The MEL1 sp directs secretion of only negligible amounts of either Echis or PAI- into the culture medium while the MForl pre-pro leader directs secretion of significant amounts of both proteins. (2) Two mutations of the MEL1 sp were constructed, ml and m3, which resulted in the secretion of both Echis and PAI- at levels comparable to the levels achieved with the MFcrl leader. Mutation ml introduces a positive charge into the n-domain plus an additional hydrophobic residue into the h-domain of the sp. Mutation m3 contains the changes introduced by mutation ml and the substitution of the helix-breaking residue Pro for Lys at position -5 relative to the signal peptidase cleavage site. (3) The secreted Echis and PAI-I are biologically active indicating that the mutant sp direct the secretion of native, correctly folded proteins. (4) The combination of mutant m3 and strain BJ1995 showed the greatest improvement of PAI- 1 secretion while mutant ml and strain JRY 188 showed the greatest improvement of Echis secretion. These data suggest that the sp/protein interaction in combination with the host strain background are important for optimal secretion and no single combination will function best for all proteins. (5) Finally, these mutant versions of the MEL1 sp may prove generally useful for directing the secretion of heterologous proteins by yeast.

111 Heukeshoven,

ACKNOWLEDGEMENTS

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fast staining in PhastSystem

We thank M. Neeper and C. Przysiecki for helpful suggestions, C. Carty, W. Herber, D. Montgomery and R. Ellis for critical reading of the manuscript, R. Lynch for Echis activity assays, P. Burke and C. Dennis-Sykes for synthesis of oligos and D. Sage1 for the careful preparation of the manuscript.

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Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast.

The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture med...
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