Efficient secretion of Bacillus amyloliquefaciens α-amylase cells by its own signal peptide from Saccharomyces cerevisiae host

161

Gene. 59(19X7)161-170 Elsevier GEN 02163

Efficient secretion of ~uc~~Z~S a~y~oli4~e~aciens Saccharomyces cerevisiae host DNA;

(Recombinant

heterologous

gene expression;

a-amylase

glycosylation;

cells by its own signal peptide from

yeast)

Peter Hackman b, P%ivi Lehtovaara b, Jonathan K.C. Knowlesb and Sirkka Keriinena

Laura Ruohonen”,

” Recombinant DNA Laboratory, University of Helsinki, SF-00380 Helsinki (Finland) and ’ Biotechnical Laboratory, VTT, SF-02150 Espoo (Finland) Tel. 358-O-45651 10 Received

2 July 1987

Accepted

29 July 1987

SUMMARY

of ~~ci~~us a~~lo~~q~efacie~ ilr-amylase was studied in yeast S~cc~ffro~_yce~~ was removed by BAL 3 1 digestion and three forms of the sc-amylase gene were constructed: the Bacillus signal sequence was either complete (YEpzal), partial (YEpaa2) or missing (YEpsra3). Secretion of r-amylase into the culture medium was obtained with the complete signal sequence only. The secreted x-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated cl-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm. The

expression

and secretion

cerevt’siue. The Bacillus promoter

INTRODUCTION

The ability of the yeast S. cereWae to secrete proteins into the culture medium makes it an attractive host for the production of heterologous proteins. Glycosylation of proteins, specific for eukaryotes, is

Correspondenceto: Dr. ratory, (Finland)

University

L. Ruohonen,

of Helsinki,

Recombinant

Valimotie

‘4bbreviations:

aa, amino acid(s); A,,, bp, base pair(s);

ER, endoplasmic PMSF,

reticulum;

phenylmetbylsuIfony1 complete (Sherman

absorbance

BSA, bovine

kb, 1000 bp; fluoride;

medium (Sherman

dodecyl sulfate; TCA, trichloroacetic medium

Helsinki

Tel. (0)410566.

Ap, ampicillin;

synthetic

DNA Labo-

7, SF-00380

at 600 nm;

serum Km,

R, resistance;

albumin;

kanamycin; SC, yeast

et al., 1983); SDS, sodium acid; YPD, yeast complete

et al., 1983).

037X-l 119iX7/$03.50 0

1987 Elsevier

Science Publishers

B.V. (Biomedical

another potential advantage of yeast as compared to bacteria. The glycosylation pattern in yeast, however, differs from that of animal cells. In yeast long, branched mannose chains replace the terminal sugars of the animal cell complex type glycans (Ballou, 1982; Kornfeld and Kornfeld, 1985). Therefore it is of interest to study the effects of yeast type glycosylation on secretion and biological activity of heterologous proteins. To study this question, we have chosen to investigate the expression of B. a~~vloliquefa~~ens cr-amylase in S. cerevisiae. In its original host cr-amylase is efficiently secreted into the culture medium (Ingle and Erickson, 1978). As a bacterial enzyme a-amylase is not glycosyIated but it contains four potential N-glycosylation sites (Takkinen et al., 1983). Like in many proteins secreted by Bacillus the signal peptide Division)

162

LiCl-procedure

of a-amylase is unusually long, 31 aa, compared to those typically found in eukaryotes which are around

transformations.

20 aa long. Here we describe

(c) Preparation

the synthesis,

processing

and

efficient secretion of B. amyloliqutlfacietw sc-amylase by 5’. cerevisiue from cloned DNA on a multicopy plasmid.

The secretion

was guided

by the Bucillus

signal peptide and the enzyme became glycosylated. The glycosylated

x-amylase

retained

its enzymatic

activity.

of Ito et al. (1983) was used for yeast

of yeast cell and spberoplast

tracts Only early to middle logarithmic-phase used. Immunoblotting and lysed with 22, containing trasylol

cells were

cells were collected

SDS (400 ~111-2 x 10’ cells)

(Bayer).

The lysates

were vigorously

vor-

of glass beads (diameter

0.45

mm) for 3 min and then boiled for 3 min. Vortexing and boiling were repeated. a-Amylase activity samples: cells (5-10 x 10’) were pelleted and resuspended in 1 ml of 1.2 M sorbitol, 0.1 M Tris . HCl, pH 7.5 containing 50-100 pg of zymolyase 1OOT

AND METHODS

(a) Strains and media Escherichiu coli was a derivative of HBlOl and was grown in Luria broth (Lennox, 1955) containing 100 pg Ap/ml. Luria plates were supplemented with 1.5y0 agar. 5’. cerevisiue strains were DBY746 (a his3Al leu2-3 leu2-112 ~23-52 trpl-289 cyhR) from David Botstein and secl (a secl-1 his4 leu2-3 leu2-112 trpl-289 uru3-52) and sec7 (a sec7-1 his4 leu2-3 feu2-112 trpl-289 uru3-52) from Randy Schekman. The yeast media (YPD and SC) were prepared according to Sherman et al. (1983). SC-Leu medium was lacking leucine. The yeast plates were supplemented with 27; agar. (b) Plasmid constructions, transformations

samples:

1.5 mM PMSF (Sigma) and 100 units/ml

texed in the presence

MATERIALS

ex-

DNA preparations

and

Standard recombinant DNA methods were used (Maniatis et al., 1982). DNA fragments were isolated from 1% agarose gels by binding to a DEAE membrane (NA45; Schleicher & Schuell) and eluting with 1.5 M NaCl. Nucleotide sequences were determined by the dideoxy method (Sanger et al., 1977). Large-scale plasmid DNA from E. coli was isolated after amplification with chloramphenicol (180 pg/ml; Clewell, 1972) by the cleared lysate method of Clewell and Helinski (1969). Small-scale plasmid DNA from E. coli was prepared by the alkaline extraction procedure (Birnboim and Doiy, 1979). Small-scale plasmid DNA isolation from S. cerevisiue was as described by Keranen (1986). E. coli was transformed according to Hanahan (1983). The

(Seikagu Koguo Co., Ltd.) and incubated with gentle shaking at 37°C for lo-20 min until 95 to lOOo/, became spheroplasts. The spheroplasts were harvested, washed once with 1 ml of 1.2 M sorbitol, 0.1 M Tris HCI, pH 7.5, and lysed in water (l-2 x lo7 spheroplasts/ml).

(d) a-Amylase

activity assays

Plate assay: individual yeast colonies were grown on SC-Leu plates covered with 6 ml of agar containing 1 y,, starch for three days at 30°C and stained with 10 mM 12, 10 mM KI solution. Quantitation assay: spheroplast extract or culture medium was tested for z-amylase activity using the Phadebas@ amylase test (Pharmacia Diagnostics). Incubation was for 4 h at 37 ^ C with vigorous shaking. Units of x-amylase were estimated by comparison with commercial Bacillus a-amylase preparation (Sigma; EC 3.2.1.1) where 1 unit of r-amylase liberates 1.O mg of maltose from starch in 3 min at 20°C pH 6.9.

(e) Immunoblotting The proteins in yeast-cell extract or in culture medium were immunoblotted as described by Keranen (1986) using polyclonal antiserum against Bacillus a-amylase (Sigma; EC ‘3.2.1.1). Immunoreactive material was detected by autoradiography of ‘2sI-labelled protein A (Amersham) on x-ray film (Kodak X-Omat 5).

163

d ClaI BAL 31

\1

d BamH

I

\1 EcoRI

BamHI+

methylase

Sal I /-

Q

d

BamHI+

Fig. I. Construction

ofrecombinant

plasmids

with a-amylase

1982) was cut at a unique CIaI site and digested to protect

an internal

cleavage

isolated.

Transformants

The YRpaa

in YEpaa3 Restriction and thick

with BamHI

and HindIII.

cross-hatched

enzyme

cleavage DNA.

box ADCI

in YEpza2

fragments YEpaa2

as compared

sites are: B, BumHI; The black

terminator

(T).

to pAAH

The restriction

The BarnHI

linearized

with the a-amylase

were moved

to obtain the plasmids

and the opposite lines yeast

a plasmid

cassettes

into E. coli to obtain plasmid YEpaal. before ligation to pAAH

gene. To eliminate

ligase

the bacterial

with BAL 31 for 20 min, then digested

were ligated with pAAR6

carrying

expression

and Sal1 and pAAH

\DNA

site. Sticky ends were filled in with the Klenow

linkers were added and the fragments to Ap resistance.

_J

HindId

fragments.

digests were combined

C, ClaI; E, EcoRI; the a-amylase

I (PolIk),

gene insert;

plasmid

(YRpaa)

was digested

were isolated by 1 y0 agarose of the a-amylase

with blackened

H, HindIII;

YRpaal

for transcription

EcoRI E. coli were

with BumHI

and ligated with T4 DNA ligase and transformed

and YRpaa3

The orientation

Arrows

orientation

(Palva,

with EcoRI methylase

of E. co/i DNA polymerase

gene insert in correct

from YRpaa2

to YEporal.

box indicates

fragment

region plasmid pKTHl0 and treated

with EcoRI. The ligation mixture was used to transform

as BarnHI

and YEpaa3.

promoter

with BarnHI

heads indicate

expression

gel electrophoresis cassette

the direction

M, MboI. Thin lines show bacterial the hatched

box ADCI

promoter

is the same

of transcription. plasmid

DNA

(P), and the

YRpaa3

--\-------_ ___I_._

__.._^“-~---

-M

--------

----M

CA TCA GiX’ G/A AA f&-C

AL?.? CTGEG

ZAG TAT

Fig. 2. Nucleotide sequence betweenADC1 promoter and r-amylase gene determined for three YRpxa plasmids. Six nucleotides from the 3’ end ofADCI promoter(P) are shown followed by the L?coRl linker (underlined). The cl-amylase nucleotide sequence is shown in italics, typed without spacing far the promoter (P) and in triplets for the coding region. The first ATG codon in each insert is underhncd and the proposed N-terminal sequences for different yeast-produced z-amylases are shown. Numbers -31 and + 1indicate the N termini for the bacterial z-amyiase signal peptide and for mature z-amyfase, respectively. Hydrophobic amino acids are underfined, positively charged amino acids are shown in italics. YRprat-YRpaa3, yeast replicating plasmids containing the origin of replication from yeast chromosomal DNA and the x-amylase gene with either complete (I), partial (2) or missing (3) signal sequence; see RESULTS AND DISCUSSION, section a, and Fig. 1 (YRpaal).

AAiCAAiX3MJEG

165

RESULTS

AND

gradation

DISCUSSION

staining (a) Construction The

of recombinant

x-amylase

contained (Palva,

gene

in a 2.3-kb

insert

starch

in plasmid

is

pKTH10

region of the cc-amylase

by

a-amylase

were seen only around

the a-amylase

of B. amyloliquefaciens

1982). The promoter

gene was removed

plasmids

of

colonies

gene with the complete

quence.

This suggested

that

secreted

into the culture

medium.

The a-amylase

contains

after

iodine

containing signal

se-

cc-amylase was being

four potential

lation sites. Thus ifthe a-amylase

N-glycosy-

present in the yeast

by BAL 31 digestion

and frag-

culture medium

ments carrying the gene, now heterologous

at their 5’

suggest that the enzyme is secreted through the yeast

ends

were ligated

pAAR6

to the yeast

(Ammerer,

YRpaa plasmids Ap resistance.

expression

1983) (Fig. 1). The

were used to transform

vector resulting E. coli to

The exact junction (Fig. 2) between the yeast ADCI promoter and the cc-amylase gene insert was determined by nucleotide sequencing of the clones. Recombinant plasmids with three different junction sites were selected for study. One insert started 12 bp upstream from the initiating ATG, so leaving the signal sequence intact (YRpaal), the second lacked the first 16 bp of the signal sequence (YRpcra2) and the third began 8 bp upstream from the coding region of mature cc-amylase (YRpaa3). Transcription of the cr-amylase gene in these plasmids should initiate at the ADCI promoter but the translation start signals must be provided by the gene itself. If translation initiation occurs at the first ATG codon (underlined in Fig. 2) present in each insert, cc-amylase with a complete signal peptide of 31 aa (YRpaal), (YRpaa2)

with a partial signal peptide of only 16 aa and mature a-amylase lacking its first 5 aa

(YRpsta3) will be synthesized (Palva et al., 1981). When the three plasmids described above were transformed into S. cerevisiae only low levels of a-amylase activity were detected. The transformants were found to be very unstable even under selective conditions. For this reason, the a-amylase expression cassettes of pAAR6, in which the origin of replication comes from yeast chromosomal DNA, were moved to pAAH (Ammerer, 1983) containing the origin of replication from the yeast 2~ plasmid (Fig. 1). The more stable plasmid constructions YEpclal-YEpcta3 were used in further experiments. (b) Secretion and glycosylation lase in yeast

of Bacillus

c+amy-

When yeast transformants were grown on starchcontaining plates, colourless haloes indicating de-

was glycosylated,

secretory

pathway.

blotting.

Tunicamycin

it would strongly

This was studied

by immuno-

was used to prevent potential

N-glycosylation (Mahoney and Duksin, 1979). The immunoreactive material precipitated from the culture medium in the absence of tunicamycin was seen in three distinct bands all migrating more slowly than the cc-amylase from Bacillus (Fig. 3, lane 3). In the presence of tunicamycin only one band was detected authentic

migrating at the same position as the Bacillus a-amylase (Fig. 3, lane 4). This

demonstrates that the cc-amylase secreted from yeast cells became N-glycosylated. Based on the A4, estimations, the three glycosylated forms of r-amylase could represent molecules containing 2, 3 or 4 yeast type core glycan but not the long mannose outer chains typical of yeast (Ballou, 1982). We suggest that the complete Bacihs signal peptide was synthesized and was able to lead to secretion in yeast. Furthermore, glycosylation was not required for efficient secretion of cl-amylase. The normal size of the unglycosylated z-amylase secreted in the presence of tunicamycin suggests that the signal peptide was cleaved off. Recently, similar results were reported for human salivary r-amylase in yeast (Nakamura

et al., 1986; Sato et al., 1986).

(c) Bacillus a-amylase is secreted through normal yeast secretory pathway Glycosylation that the protein

of the cc-amylase strongly syggests had at least entered the ER in the

yeast secretory route. However, since the yeast-type outer-chain addition was not observed, it is not clear whether the cr-amylase traversed the later steps ofthe normal secretory pathway. To study this YEpaal was transformed into sec7 and secl strains. In these strains secretion of proteins is blocked at 37°C so that the proteins accumulate at Golgi and in secretory vesicles, respectively (Novick et al., 1980; 1981). As shown in Fig. 4 secretion of z-amylase into the

166

l.O-

92.5 69

46

30 12345 Fig. 3. Secretion carrying

and processing

yeast cells (DBY746)

0.5 were transferred

of z-amylase

into fresh medium

absence (lanes I and 3)

and proteins

for I20 min. Cells were removed

separated

trophoresis

for

presence

with IO”,, TCA and washed by 7.5-15’3, 16 h with

descrihcd

constant

in MATERIALS

proteins

(Amersham)

precipitated

used

current METHODS,

the kDa

plasmid

yeast 2~1plasmid

Bncihs

containing

(I2

mA) in the

Culture

medium

pAAH5. YEpaal,

AND

vector

DISCUSSION.

yeast

from the

and the cc-amylase gcnc with complete

see RESULTS

as

section e. The

the origin of replication

LlJ 5

1

signal

section a. and

10

Time (h)

gel elec-

(lanes 3 and 4). Lane

r-amylase.

LLtrttt

BSA

once with -20°C

for the “C-methylated

as M, markers.

1 and 2) or YEpzal

5. IO ng of commercial

sequence;

by centrifugation

polyacrylamide

from yeast strain DBY746 carrying

alone (lanes

0.1 -

and the growth

1970) and immunoblottcd

AND

on the left specify

episomal

gradient

of 0. I ‘II, SDS (Laemmli,

numbers

plasmid

was changed

in 9 ml of culture medium with 20 pg of carrier

were precipitated acetone,

and grown further in the

or presence (lanes 2 and 4) oftunicamycin

(10 ktg,‘ml). At 60 min the medium was continued

in yeast. Plasmid-

grown at 30°C in SC-Leu to A,,,,,

Fig.4.

Secretion

of cc-amylasc

YEpral-carrying at26’CtoA Growth

(,,r,)0.15. pelleted and transferred

was continued

(filled symbols). were removed into

the

by .recl

and

src7

mutants.

yeast cells (seal or sec7) wcrc grown in SC-Leu

Aliquots

wcrc taken

by centrifugation

culture

MATERIALS

medium

AND

at 60-min intervals,

cells

and cc-amylasc activity secreted

was

METHODS,

say). Circles, .scc/ cells transformed cells transformed

into fresh medium.

either at 26‘C (open symbols) or at 37’C

determined

as described

section d (quantitation with YEpatal;

squares,

in as.sec7

with YEpral.

Fig. I.

(d) Partial a-amylase signal peptide is not functional in yeast

culture medium at restrictive temperature was blocked in both mutants, while the secretion at permissive temperature occurred normally. Thus we conclude that Bacillus a-amylase is, indeed, secreted in yeast through the normal secretory pathway from ER to Golgi and via secretory vesicles to cell surface (Schekman, 1982).

The intracellular cc-amylase from yeast transformants containing the three different forms of the sc-amylase gene is shown in Fig. 5. In transformants containing the complete signal sequence the intracellular z-amylase was glycosylated (lane X), while in the other transformants either lacking the signal sequence (lanes 2 and 3), or containing the

167

partial

signal sequence

glycosylated

a-amylase

(lanes

5 and 6) only non-

was detected.

When the signal sequence

was lacking, the size of

the !x-amylase was slightly smaller than that of the authentic a-amylase most probably due to the lack of 5 aa from its N terminus (Fig. 2). The size of a-amylase from the partial signal sequence construction suggests that a partial signal peptide was synthesized but not cleaved off. This would be the case if the protein remained

in the cytoplasm.

These results

indicate that the partial signal peptide of 16 aa is not sufficient

for secretion

in yeast.

(e) Complete Bacillus signal peptide allows efficient secretion of a-amylase by yeast The efficiency of z-amylase secretion was studied by measuring the cc-amylase activity both from culture medium and from spheroplast lysates. Activity in the culture medium was detected only with transformants containing the E-amylase gene with the complete signal sequence (Table I). Secretion was very efficient, since the z-amylase activity secreted during 2 h represented 75% of the total activity.

12345678 Fig. 5. Intracellular cells (DBY746)

r-amylase

5, 8) or prcsencc

(lanes

Fig. 3, harvested then

Based on the enzymatic activity compared with the bacterial enzyme, the amount of z-amylase secreted under these conditions is about 10 pg/liter/h. However, in transformants containing the a-amylase gene with the truncated signal sequence close to lOOo/, of cc-amylase activity was intracellular.

cell

yeast-cell

were

section

extracts

prepared

c; immunoblotting

see

were: 50 ng (lanes

I, 2,

as described

for

(MATERIALS samples). AND

of yeast protein

AND

Proteins

in

for Fig. 3 and METHODS,

loaded onto the gel

I, 2, 3); 35 ug (lanes 5,6); 25 keg (lane 8). Cell

from yeast transformed

(lanes 2 and 3); YEpra2 from DBY746

yeast

(lanes

once with water and

as described

MATERIALS

section e. The total amounts

4 and 7,5 ng ofBacii/us TABLE

washed

were separated

immunoblotted;

extract

Plasmid-carrying

3, 6) of tunicamycin

by centrifugation,

extracts

METHODS,

in yeast.

were grown at 3O’C in the absence

with pAAH

(lane I); YEpza3

(lanes 5 and 6); YEpzal z-amylase

transformed

(lane X). Lanes

and 30 pg of yeast cell protein

with pAAH5.

1

r-Amylase

activity

in yeast

Plasmid

Signal sequence

Sample

z-Amylase

activity

(units x 10e5 per 4 x 10’ cells)” YEpral

complete

partial

Y Epra2

I’ Plasmid-carrying

METHODS,

in medium

2033

(75)

intracellular

690

(25)

in medium

24

(2)

intracellular

958

(98)

in medium

UD

intracellular

103

yeast DBY746 cells (5 x 10’) were grown as described

was collected.

AND METHODS, plasmid.

(16 aa)

absent

YEpza3

medium

(3 1 aa)

The cells were washed

section E (z-amylase

section d (quantitation

for Fig. 3. Cells were removed

once with water and spheroplast

activity

samples).

z-Amylase

assay). UD, undetectable.

Numbers

(100)

activities

extracts

were prepared

were determined

in parentheses

by centrifugation as described

as described

and the culture in MATERIALS

in MATERIALS

show the % of total z-amylase

AND

activity for each

The total x-amylase

activity in YEpcla2 transfor-

mants was 36% of that of YEpclal transformants. Low-level intracellular activity only was detected in

(g) Conclusions To our knowledge

the work reported

here is the

YEpaa3 transformants. At the moment, it is not possible to say whether this reflects lower expression

first in which a prokaryotic signal sequence is shown to lead to genuine secretion in yeast. The signal

levels or de~adation of the protein addition, a-amylase with an uncleaved

peptide was cleaved off correctly or very close to the authentic cleavage site. A number of eukaryotic

peptide

or missing

5 aa from its N terminus

could

activity.

(f) Effects of a-amylase

expression

times and stability

1985; Wood et al., 1985; Nakamura et al., 1986). Most detailed characterization was carried out for

of yeast transformants

Generation

Plasmid”

Stability”

time h

(min)

i

(“0)

180

26

YEpcla’

144

46

YEpra3

14X

56

pAAH

147

58

YEpza

” Plasmid-c~irryin~yeast pAc\HS; Fig.

strain was DBY746. YEpaal-YEpaa3,

see RESULTS

AND

DISCUSSION,

section a, and

I (YEpaal).

’ Plasxnid-carrying

yeast

selective conditions

for 15.5 h. Samples

cells

were

grown

at

3O’C

under

were taken at 2-h inter-

vals and cell concentration

was determined

The growth curves obtained

were used to estimate

by measuring

A6,,rj.

the generation

times. ‘ Pl~lsmid-carrying YPD medium, incubated growing plates.

yeast cells were grown

at 30°C for 27 h in

then plated on both YPD and SC-Leu plates and

at 30°C until colonies on SC-Leu

plates

appeared.

as compared

Mean of four determinatiolls.

been

of yeast trans-

II time and stability

have previously

et al., 1984; Innis et al., 1985; Oberto and Davison,

stability of YEpcla2 transformants was somewhat reduced. The YEpaal tr~sform~ts, efficiently secreting a-amylase, had a clearly increased generation time and also lost the plasmid most frequently. Thus, the expression and/or the secretion of x-amylase is slightly deleterious to the yeast cells.

Generation

signal peptides

on yeast ceils

formants expressing the three different forms of the !x-amylase gene are shown in Table II. Expression of the intracellular forms of a-amylase had no effect on the generation time as compared to the transformants containing the vector alone. However, the

TABLE

heterologous

reported to function in yeast leading to secretion (Hitzeman et al., 1983; Thomsen, 1983; Rothstein

have lowered enzymatic

The generation

or both. In partial signal

Stability, to colonies

‘+,,,colonies un YPD

the human a-amylase secreted by yeast and it was shown to be processed and modified in the same manner as in animal cells (Sat0 et al., 19%). The reason why the partial cc-amylase signal peptide did not function is not obvious. As recently demonstrated (Kaiser et al., 19871, the requirements for the signal peptide function in yeast seem not to be as stringent as some previous results have suggested (discussed by Schekman, 1985). The predicted partial signal peptide of a-amylase very much resembles those known from the yeast proteins (Kurjan and Herskowitz, 1982; Singh et al., 1983; Taussig and Carlson, 1983; Bajwa et al., 1984; Bostian et al., 1984; Liljestrbm, 1985; Yamashita, 1985). They all are very hydrophobic and mostly contain just one positively charged amino acid either at their N terminus or C terminus. However, other factors such as c~?nformation of the pre-amylase may also be involved in the observed requirement for the complete signal peptide. Evidently the number of charged residues at the N terminus is not critical since some yeast proteins contain none (Taussig and Carlsson, 1983; Liljestrom, 1985) and we have shown here that as many as four in Bncillus or-amylase are tolerated. It was interesting to observe that the presence of the (core) glycans did not abolish the enzymatic activity of a-amylase. This shows that glycosylation as such is not necessarily deleterious to proteins that lack sugar chains in their natural form. The r-amylase was secreted through the normal secretory pathway as demonstrated by its glycosyIation and behaviour in set mutants. Although the secreted a-amylase was transported to the cell surface via the Golgi complex and the secretory vesicles it did not become hypermannosylated like the secretory proteins of yeast itself. Similar findings have

169

been reported (Innis (Sato

for Aspergillus awamori glucoamylase

et al., 1985) and human

salivary

cr-amylase

et al., 1986) which were ~-glycosylat~

and

secreted by yeast but did not contain the yeastspecific outer sugar chains. One explanation for this phenomenon

is that core glycosylation

ER, perhaps assume

before

the protein

occurs in the

has had

its final conformation.

time to

The core carbohy-

drates, however, might no longer be accessible after protein folding. Since certain glycoproteins can be

cd’ in the presence

of chloramphenicol.

Clewell,

D.B. and He&ski,

protein

conversion

to an open circular

Hanahan,

D.: Studies

plasmids.

of

other

tion of certain

heterologous

Hitzeman,

DNA-

and induced

DNA form. Proc. Natl. Acad. of Escherichia coli with

on transformation

R.A., Leung, D.W., Perry, J., Kohr, W.J., Levine, H.L.

and Goeddel, Science

D.V.: Secretion

of human interferons

by yeast.

219 (1983) 620-625.

Microbial.

R.J.: Bacterial

a-amylases.

Adv. Appl.

24 (1978) 257-278.

V.P.,Tal,

M.J., McCabe,

P.C., Cole, G.E., Wittman,

R., Watt, K.W.K., Gelfand,

Meade,

glycoproteins.

circular

J. Mol. Biol. 166 (1983) 557-580.

Innis, M.A., Holland,

glycans

yeast could be a useful host for produc-

110

Sci. USA 62 (1969) 1159-l 166.

to

high-mannose-type

D.R.: Supercoiled

in Escherichia coli: purification

complex

Ingle, M.B. and Erickson,

the

J. Bacterial.

( 1972) 667-676.

secreted by yeast without becoming hypermannosylated and since the yeast core glycans are very similar eukaryotes,

in Escherichia

Clewell, D.B.: Nature ofCo1 El plasmid replication

J.H.: Expression,

D.H., Holland,

glycosylation,

J.P. and

and secretion

of an

by Saccharomyces cerevisiae. Science

Aspergillus glucoamylase 228 (1985) 21-26. Ito, H., Fukuda,

Y., Murata.

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