Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a...

Protein Disulfide Isomerase, But Not BindingProtein, Overexpression Enhances Secretionof a Non-Disulfide-Bonded Protein in Yeast

Jason D. Smith, Benjamin C. Tang, Anne Skaja Robinson

Department of Chemical Engineering, University of Delaware, 259 ColburnLaboratory, Newark, Delaware 19716; telephone: 302-831-0557; fax: 302-831-6262; e-mail: [email protected]

Received 5 May 2003; accepted 21 August 2003

Published online 31 December 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10853

Abstract: In eukaryotes, secretory proteins are folded andassembled in the endoplasmic reticulum (ER). Many hete-rologous proteins are retained in the ER due to suboptimalfolding conditions. We previously reported that heterolo-gous secretion of Pyrococcus furiosus h-glucosidase inSaccharomyces cerevisiae resulted in the accumulation of alarge fraction of inactive h-glucosidase in the ER. In thiswork, we determine the effect of introducing additionalgenes of ER-resident yeast proteins, Kar2p (binding protein[BiP]) and protein disulfide isomerase (PDI), on relieving thisbottleneck. Single-copy expression of BiP and PDI workedsynergistically to improve secretion by f60%. In an effortto optimize BiP and PDI interactions, we created a library ofh-glucosidase expression strains that incorporated fourcombinations of constitutively or inducibly-expressed BiPand PDI genes integrated to random gene copynumbers inthe yeast chromosome. Approximately 15% of the trans-formants screened had secretion level improvements higherthan that seenwith single BiP/PDI gene overexpression, andthe highest secreting strain had threefold higher h-glucosi-dase levels than the control. Nineteen of the improvedstrains were re-examined for h-glucosidase secretion aswell as BiP and PDI levels. Within the improved trans-formants BiP and PDI levels ranged sevenfold and tenfoldover the control, respectively. Interestingly, increasing BiPlevels decreased h-glucosidase secretion, whereas increas-ing PDI levels increased h-glucosidase secretion. The actionof PDI was unexpected because h-glucosidase is not adisulfide-bonded protein. We suggest that PDI may beacting in a chaperone-like capacity or possibly creatingmixed disulfides with the h-glucosidase’s lone cysteineresidue during the folding and assembly process. B 2004Wiley Periodicals, Inc.

Keywords: Archaea; binding protein (BiP); protein disulfideisomerase (PDI); Pyrococcus furiosus; Saccharomycescerevisiae; optimization; secretory pathway; endoplasmicreticulum; h-glucosidase; chaperone; Hsp70

INTRODUCTION

Overexpression of a foreign protein in any recombinant

host can lead to misfolding of the foreign protein due to

non-native, suboptimal conditions and/or overloading of

the host’s machinery. Gene copy optimization is a typical

strategy used to optimize heterologous protein expression.

However, in the case of secreted proteins, this strategy can

lead to saturation or overloading of the secretory pathway,

typically in the endoplasmic reticulum (ER) (Parekh et al.,

1995; Parekh and Wittrup, 1997; Robinson et al., 1994;

Shusta et al., 1998).

By varying gene copynumber, Parekh and Wittrup (1997)

reached 180 mg/L of secreted BPTI in Saccharomyces

cerevisiae. They found that BPTI accumulated in the ER in

an unfolded form, which they believed was due to dilution of

the necessary folding machinery. Robinson and Wittrup

(1995) showed that overexpression of heterologous proteins

in S. cerevisiae led to decreased soluble levels of ER-

resident immunoglobulin heavy-chain binding protein (BiP)

and protein disulfide isomerase (PDI). Consequently, it has

been shown that reduction of BiP levels resulted in decreased

heterologous protein secretion (Robinson et al., 1996). BiP

and PDI, among others, participate directly in the active

folding of proteins in the ER and only properly folded

proteins are allowed to exit the ER. Therefore, manipulation

of the ER folding environment may produce further im-

provements in S. cerevisiae’s secretory productivity.

BiP and PDI are commonly studied, abundant ER-resident

proteins. BiP, also known as Kar2p in yeast, is an Hsp70-

class heat shock protein (Haas and Wabl, 1983) that

functions in protein folding as well as being an important

part of the cell’s translocation machinery (Matlack et al.,

1999). BiP aids in protein folding through the binding of

hydrophobic amino acid stretches in the immature protein.

Binding of these exposed stretches stabilizes the protein and

prevents it from aggregating with other unfolded/misfolded

proteins. Through ATP hydrolysis, BiP releases the bound

polypeptide allowing for continuous cycles of binding and

releasing thus giving the polypeptide a chance to bury its

B 2004 Wiley Periodicals, Inc.

Correspondence to: A. Robinson

Contract grant sponsors: DuPont (Young Investigator Award); ACS

Petroleum Research Foundation

Contract grant number: 35916-G4

exposed hydrophobic residues in the process of proper

folding (Rowling and Freedman, 1993). BiP also plays a role

in proofreading, through ER-associated degradation (Brod-

sky et al., 1999).

PDI, an abundant ER foldase (Rowling and Freedman,

1993), is an essential yeast protein that catalyzes the oxida-

tion, reduction, and isomerization of disulfide bonds (Laboi-

ssire et al., 1995). PDI also has a well-demonstrated, albeit

poorly understood, chaperone ability (Dai and Wang, 1997;

Klappa et al., 1998; LaMantia and Lennarz, 1993); that is,

there is no known specific binding site as there is with BiP.

By manipulating chaperone and foldase levels, improve-

ments in yeast/eukaryotic secretion have been found for a

number of proteins. Robinson et al. (1994) reported four- to

tenfold higher secretion yields of two heterologous proteins

in the yeast S. cerevisiae by overexpressing a single inte-

grated copy of PDI. Similarly, Shusta et al. (1998) reported

approximately twofold increases in secretion of a single-

chain antibody fragment by overexpression of either BiP or

PDI in yeast. When both PDI and BiP were co-overex-

pressed, secretion levels increased eightfold. In addition,

secreted levels of the highly disulfide-bonded thaumatin

were increased approximately fivefold upon overexpression

of protein disulfide isomerase in the filamentous fungus

Aspergillus awamori (Moralejo et al., 2001). In an

Escherichia coli cell-free translation system for single-chain

antibody fragments, the addition of PDI increased functional

antibody levels (Ryabova et al., 1997).

We had previously demonstrated heterologous secretion

of the hyperthermophilic, tetrameric Pyrococcus furiosus h-

glucosidase in Saccharomyces cerevisiae to approximately

12 mg/L (Smith and Robinson, 2002). Characterization of

h-glucosidase expression revealed an ER bottleneck, with

experimental evidence indicating misfolding of the mono-

mer. In this study we optimize the folding conditions of the

ER by manipulation of the ER-resident proteins, BiP and

PDI, to improve h-glucosidase secretion levels.

MATERIALS AND METHODS

Cell Strains

The yeast strain BJ5464 (a ura3-52 trp1 leu2D1 hisD200

pep4::HIS3 prb1D1.6R can1 GAL) was obtained from the

Yeast Genetic Stock Center (Berkeley, CA) and served as

the basis for all yeast constructs. Strain JSIY017 was ob-

tained by transformation of BJ5464 with the pITy4-hintegrating plasmid containing the secretion-engineered

Pyrococcus furiosus h-glucosidase gene (Smith and

Robinson, 2002).

Media and Growth Conditions

Yeast cells transformed with integrating plasmids were

typically grown in the rich media YPD and YPG. YPD (2%

peptone; Difco, Carlsbad, CA), with 1% yeast extract

(Difco) and 2% glucose (Fisher, Pittsburgh, PA), was used

as rich growth media and YPG (2% peptone; Difco), with

1% yeast extract (Difco) and 2% galactose (Fisher), was

used as expression media.

Minimal media was used for yeast that had been

transformed with plasmids containing auxotrophic selec-

tion markers. Growth (SD-2� SCAA) or expression (SG-

2� SCAA) minimal media contained 0.67% yeast nitrogen

base (Difco), 2% glucose or galactose, 1.47% citric acid

monohydrate (Sigma), 0.42% sodium citrate (Sigma), and

synthetic amino acid supplement (2� SCAA) (Wittrup and

Benig, 1994). Leucine, tryptophan, and uracil were added

as needed to concentrations of 400 mg/L, 40 mg/L, and

40 mg/L, respectively.

Transformants were typically grown for 48 h in glucose-

based media at 30jC, 275 rpm. Five optical density (OD)-

milliliter equivalents were isolated by centrifugation (2000� g, 5 min) and the supernatant removed. Cell pellets were

resuspended in 5 mL of galactose-based media and ex-

pressed for 24 h for YPG or 48 h for SG-2� SCAA at 30jC,

275 rpm. Supernatant and cell fractions were isolated for

further analysis.

Plasmids

A CEN-ARS vector with a URA3 metabolic marker, pCT37,

was used for galactose-induced expression of yeast PDI

(Tachibana and Stevens, 1992). Similarly, pGalKar2-LEU

is a CEN-ARS vector containing a galactose-inducible yeast

KAR2 gene and a LEU2 metabolic marker (Robinson and

Wittrup, 1995).

Integrating plasmids were constructed using the pITy

integrating plasmid (Parekh et al., 1996). The plasmid

pITy4-h (Smith and Robinson, 2002) was previously used to

achieve optimal expression of h-glucosidase. A 64-bp

polylinker containing the restriction sites (from 5V to 3V)KpnI, AatII, EcoRI, EagI, BamHI, XbaI, and SacI was

purchased from Operon and was ligated into pITy4-h, which

had a KpnI–SacI expression cassette that had been removed

through restriction digestion, resulting in plasmid pITypL.

Sequential EagI and AatII digests were performed on

pGalKar2-LEU to remove the GAL1 promoter-KAR2

expression cassette (including KAR2’s transcription termi-

nator). The expression cassette was ligated into pITypL

digested with AatII and EagI, thus creating plasmid pITy-

GAL-KAR2. In a similar manner, pGAPDH-KAR2 (Rob-

inson et al., 1996) was digested sequentially with EagI and

AatII to remove the GAPDH promoter-KAR2 expression

cassette. Subsequent ligation with pITypL digested with

AatII and EagI created pITyGAPDH-KAR2.

The PDI gene was amplified from pCT37 using

polymerase chain reaction (PCR). The primer 5V-GCTCGAGATGAAGTTTTCTGCTGGTGC-3V was used

to create an XhoI site 5V of the start codon, and the primer

5V-CGGTCTAGATTTTCTCTTTCCCTAAGGCG-3V was

used to create an XbaI site 170 bp downstream of the

PDI stop codon. Both pITyGAL-KAR2 and pITyGAPDH-

SMITH ET AL.: PDI OVEREXPRESSION ENHANCES SECRETION OF YEAST PROTEIN 341

KAR2 were digested with SalI and XbaI (partial) to remove

the KAR2 gene. Subsequent ligation with the PCR-ampli-

fied gene product digested with XhoI and XbaI resulted in

integrating vectors with PDI under control of the GAL1

inducible promoter (pITyGAL-PDI) and the GAPDH

constitutive promoter (pITyGAPDH-PDI).

Integration into the genome was facilitated through

linearization of pITyGAL-KAR2 and pITyGAPDH-KAR2

with BsaBI and pITyGAL-PDI and pITyGAPDH-PDI with

XhoI before transformation into BJ5464. Transformant

colonies were selected on the basis of resistance to G418

(Sigma Co., St. Louis, MO) as previously described (Parekh

et al., 1996). Multiple pools of transformants (f20 to

30 transformant colonies each) from independent trans-

formations were transformed with pRS314-h, a CEN-ARS

pRS314 vector (ATCC, Manassas, VA) containing a TRP1

metabolic marker and a galactose-inducible P. furiosus

h-glucosidase modified for yeast secretion (Smith and

Robinson, 2002). Transformants were selected on the basis

of growth on minimal media lacking tryptophan.

Immunodetection

Cell extracts were typically prepared as described by

Robinson et al. (1996). For Western blots, cell extracts and

culture supernatants were first separated by sodium

dodecylsulfate–polyacrylamide gel electrophoresis (SDS-

PAGE). Protein transfer to nitrocellulose and Western blot

protocol were performed as previously described (Robinson

et al., 1996). Anti-BiP polyclonal primary antibody (a gift

from Jeff Brodsky, University of Pittsburgh) and anti-PDI

polyclonal primary antibody (Stressgen, San Diego, CA)

were used at 1:10,000 and 1:1000 dilutions, respectively.

Goat anti-rabbit secondary antibody conjugated to horse-

radish peroxidase (Amersham Biosciences, Piscataway, NJ)

was used for BiP and PDI detection at a 1:2000 dilution.

Antibody–antigen complexes were visualized with ECL

Plus detection reagent (Amersham) and Western blots were

imaged using the fluorimager (Model 595, Amersham).

Images were quantitated by densitometric analysis using

NIH Image 1.44 software (NIH, Bethesda, MD).

B-Glucosidase Activity Assay

The colorimetric assay used to determine h-glucosidase

activity was modified from a previously described protocol

(Wood and Bhat, 1998). Kinetic assays were performed at

90jC on a spectrophotometer (Model DU600, Beckman,

Fullerton, CA) at an analytical wavelength of 405 nm.

Quartz cuvettes (Starna Cells, Atascadero, CA) containing

0.1 M sodium acetate, pH 5.0 (capped tightly with Teflon

stoppers), were equilibrated to 90jC, after which 30 AL of

20 mM p-nitrophenol-h-D-glucopyranoside (pNPG; Sigma)

and 10 AL of isolated culture supernatant were added (1 mL

total volume). Runs were immediately initiated and

measurements were recorded every 30 s and compared

with a buffer blank. Autocleavage rates of pNPG were

undetectable during the time course of the experiments.

Activities are reported as the maximum rate of pNPG

disappearance normalized by the culture optical density.

Activities measured from cultures derived from air

shakers were typically two- to threefold lower than those

derived from water-bath shaker cultures; however, final

culture optical densities were not significantly affected.

RESULTS

Single BiP/PDI Gene Dose Results

Previously, a Pyroccocus furiosus h-glucosidase trans-

formant of the wild-type BJ5464 Saccharomyces cerevisiae

was isolated (Smith and Robinson, 2002). This trans-

formant, JSIY017, retained high levels of intracellular

h-glucosidase in the ER. The first approach to improving

h-glucosidase folding and secretion through ER manipu-

lation involved the addition of a single copy of BiP or PDI,

alone or in combination. To do this, JSIY017 was

transformed individually and in combination with pCT37

and pGalKar2-LEU, CEN-ARS single-copy plasmids con-

taining galactose-inducible PDI and BiP, respectively.

Empty CEN-ARS plasmids pRS315 and pRS316 (ATCC,

Manassas, VA) containing LEU2 and URA3 metabolic

markers, respectively, were transformed into JSIY017 as

needed to maintain identical metabolic requirements (leu�,

ura�, trp+). Cells were grown and expressed as described in

Materials and Methods. Cell fractions isolated from both

growth and expression media were assayed for BiP and PDI

levels using Western blots (see Materials and Methods). h-

Glucosidase levels in culture supernatant fractions were

determined by activity assays (Materials and Methods) and

are listed in Table I.

Individual overexpression of PDI and BiP resulted in

modest h-glucosidase secretion improvements of approx-

imately 25% and 20%, respectively, over the control

(JSIY017 transformed with pRS315 and pRS316). Co-

overexpression of both BiP and PDI gave a synergistic

effect, resulting in approximately 60% increased extra-

cellular h-glucosidase. Although secretion levels were

increased, intracellular h-glucosidase levels remained es-

sentially unchanged as measured by Western blot analysis

(data not shown), indicating that co-overexpression of ER

chaperones and foldases failed to completely remove the

ER bottleneck. All transformants had approximately iden-

tical growth characteristics in both growth and expression

media; therefore, the metabolic requirements of the cells

were likely equivalent and thus did not impact our results.

In expressing cells, BiP levels were practically unchanged

from the control when BiP, PDI, or both were overexpressed.

In contrast, BiP levels increased twofold when BiP was

overexpressed in BJ5464 parental cells (Table I). PDI

overexpression led to an approximately twofold increase in

PDI levels over the control case when either PDI or both PDI

and BiP were overexpressed.

342 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 85, NO. 3, FEBRUARY 5, 2004

A significant observation was made when BiP and PDI

levels from expressing cells were compared with those

levels in cells from nonexpressing growth media. In JSIY017

cells, BiP levels increased approximately fourfold and PDI

levels threefold in expressing cells compared with cells

grown in nonexpression (SD-2� SCAA) media (Table I).

The same result was seen for JSIY017 cells in rich media

(data not shown). In contrast, BiP and PDI levels were

approximately equal for parental BJ5464 control cells from

both growth and expression media. This suggests that h-

glucosidase expression induces significant stress on the ER

and the cell may be responding by flooding the ER with

additional chaperones and foldases through the unfolded

protein response (Chapman et al., 1998; Mori et al., 1998).

Multicopy BiP/PDI Strain Construction

Due to the modest improvement in h-glucosidase secretion

found from overexpression of single gene doses of galactose-

inducible BiP and PDI we sought to optimize the effects of

BiP and PDI on h-glucosidase secretion. Direct control of

BiP or PDI levels in vivo is difficult; therefore, we varied BiP

and PDI levels in the cell by integrating BiP and PDI genes

randomly into yeast chromosomal y-sequences using the

pITy integrating plasmid (Parekh et al., 1996). We also

varied BiP and PDI expression modes by creating plasmids

wherein BiP and PDI were under the control of either the

galactose-inducible GAL1 promoter or the constitutive

GAPDH promoter.

To test the functionality of each constructed BiP and PDI

plasmid, each of the four integrating vectors was individ-

ually transformed into the parental cell line BJ5464. Very

few pITyGAPDH-PDI and no pITyGAPDH-KAR2 trans-

formants were obtained. This was possibly due to toxic

effects of PDI and BiP constitutive expression, although

this behavior has not been reported previously in the

literature. Transformants were grown in glucose-based

YPD media to saturation, transferred to galactose-based

YPG media, and grown for 24 h. BiP and PDI levels were

detected in cell samples from both YPD and YPG media by

Western blot analysis.

Induction of BiP by galactose in pITyGAL-KAR2

transformants increased BiP levels from two- to sixfold in

YPG compared with BJ5464 control cells (data not shown)

and showed tight repression of BiP in glucose-based growth

media. PDI levels in pITyGAL-PDI transformants varied

from 1.5- to fourfold over the control and demonstrated the

same tight control over expression in YPD. Constitutive

expression of PDI in pITyGAPDH-PDI transformants

consistently showed five- to sevenfold increases over the

control in both YPD and YPG (data not shown). Because

no pITyGAPDH-KAR2 transformants could be isolated, we

were unable to evaluate constitutively expressed BiP levels.

Because the h-glucosidase gene in JSIY017 cells is

chromosomally integrated to a high number using pITy-

based integrated plasmids, we sought to avoid problems

with pITy-based BiP/PDI transformant selection. Also,

possible recombination events between BiP/PDI and h-

glucosidase pITy-based plasmids could potentially alter

h-glucosidase expression in subsequent transformants.

Therefore, we transformed integrated BiP/PDI transform-

ants of BJ5464 with pRS314-h, a CEN-ARS vector

containing a TRP1 metabolic marker, and the secretion-

competent P. furiosus h-glucosidase under the control of

the galactose-inducible GAL1-10 promoter (Smith and

Robinson, 2002). Similar to JSIY017, BJ5464 cells trans-

formed with pRS314-h also exhibited an intracellular ER

bottleneck (data not shown); however, they secrete

approximately threefold less h-glucosidase than JSIY017

cells (Smith and Robinson, 2002).

Therefore, the remaining work focuses on testing the

ability of our BiP/PDI plasmid system to screen ranges of

BiP and PDI levels to determine which levels optimize h-

glucosidase folding and secretion. Once the utility of the

BiP/PDI screening system has been proven, future work

could utilize a non-pITy-based integrating h-glucosidase

Table I. Single copy BiP/PDI overexpression in JSIY017 and BJ5464.

Overexpression of:

Sample Media BiP PDI

h-glucosidase

(AM pNPG/min/OD)

Normalized

BiP

Normalized

PDI

BJ5464 SD – – — 1.0 F 0.1 1.0 F 0.1

BJ5464 SG – – 0.0 F 0.1 1.1 F 0.0 1.1 F 0.0

BJ5464 SG – + — 1.2 F 0.1 2.4 F 0.1

BJ5464 SG + – — 2.1 F 0.0 1.2 F 0.1

JSIY017 SD – – — 1.5 F 0.3 1.3 F 0.0

JSIY017 SG – – 3.2 F 0.1 3.8 F 0.3 2.7 F 0.1

JSIY017 SG – + 4.0 F 0.2 4.0 F 0.2 4.7 F 0.5

JSIY017 SG + – 3.8 F 0.5 4.0 F 0.1 2.2 F 0.4

JSIY017 SG + + 5.2 F 0.1 3.9 F 0.1 5.3 F 0.4

Secreted h-glucosidase and intracellular BiP and PDI data for each transformant in both glucose-based growth media (SD) and galactose-based

expression media (SG). BiP and PDI levels were measured by Western blot analysis (see Materials and Methods) and normalized to BiP and PDI level from

BJ5464+pRS315+pRS316 in SD media. Extracellular h-glucosidase levels were determined by activity assays (see Materials and Methods) and normalized

by culture optical density. Errors represent the standard deviation from the average of duplicate samples.


plasmid such that a high h-glucosidase gene copy number

and BiP/PDI engineering could be employed concurrently.

BJ5464 cells were transformed with various combina-

tions of BiP and PDI plasmids (galactose-inducible and

constitutive promoters) and selected for resistance to G418.

Approximately 20 to 30 colonies from independent trans-

formations were pooled together and then transformed with

pRS314-h. Transformants were then selected on minimal

media lacking tryptophan and lacking G418, because inte-

grated pITy plasmids are typically stable. This sequential,

single-selection process decreased overall population

variation due to colony pooling, but enabled transformation

with all three expression plasmids.

By transforming BJ5464 with the various combinations

of BiP (Kar2p in yeast) and PDI expression plasmids we

created a fully inducible system, two mixed inducible–

constitutive systems, and a fully constitutive system, all

containing a single gene dose of galactose-inducible h-

glucosidase. The systems were denoted as follows: (1)

GAL-KAR2+GAL-; (2) GAL-KAR2+GAPDH-PDI;

(3) GAPDH-KAR2+GAL-PDI; and (4) GAPDH-KAR2+-

GAPDH-PDI. BJ5464-C (BJ5464 transformed with

pRS314) served as the control for BiP and PDI levels and

BJ5464-h (BJ5464 transformed with pRS314-h) served as

the control for h-glucosidase levels.

Similar to the case for individual integrants of BiP and

PDI into BJ5464, low efficiencies were obtained for

combinations including constitutively expressed BiP vec-

tors, again presumably due to the possible toxic effects of

constitutive BiP overexpression. Therefore, in total, 100

transformants of each GAL-KAR2+GAL-PDI+h and

GAL-KAR2+GAPDH-PDI+h, 20 transformants of

GAPDH-KAR2+GAL-PDI+h, and 19 transformants of

GAPDH-KAR2+GAPDH-PDI+h, where h denotes the

addition of a h-glucosidase gene, were isolated and screened

for improved h-glucosidase secretion.

B-Glucosidase Screen

For all four systems, duplicate samples were grown to

saturation in 5-mL SD-2� SCAA(�trp) (abbreviated SD)

minimal media cultures, then transferred to 5-mL SG-2�SCAA(�trp) (abbreviated SG) minimal media cultures, and

then allowed to express h-glucosidase for 48 h, as described

in Materials and Methods. Culture supernatants were

isolated from SG media and assayed for h-glucosidase

activity (Fig. 1). Activities were normalized by cell optical

density and values reported are relative to BJ5464-h control.

Growth characteristics for all cultures, including the control,

were roughly equivalent (data not shown).

Figure 1. h-Glucosidase screen of the system transformants. Transformants of GAL-KAR2+GAL-PDI+h (A), GAL-KAR2+GAPDH-PDI+h (B),

GAPDH-KAR2+GAL-PDI+h (C), and GAPDH-KAR2+GAPDH-PDI+h (D) were grown in glucose-based growth media to saturation, transferred to

galactose-based media, and expressed for 48 h at 30jC. Culture supernatant samples were isolated and assayed for h-glucosidase activity (see Materials

and Methods). Activity values were normalized to BJ5464-h control cells and reported as relative h-glucosidase levels.


A significant number of transformants were found with

h-glucosidase secretion levels higher than the original BiP/

PDI single gene dose experiments. Approximately 10% of

GAL-KAR2+GAL-PDI+h, 15% of GAL-KAR2+GAPDH-

PDI+h and GAPDH-KAR2+GAPDH-PDI+h, and 65%

of GAPDH-KAR2+GAL-PDI+h transformants had im-

proved secretion levels, higher than the 60% improvement

seen for the single BiP/PDI gene overexpression case.

Strains with constitutive BiP expression showed h-glucosi-

dase improvements that were, on average, much higher than

those for inducible BiP and constitutive PDI strains. Strains

with constitutive BiP and inducible PDI expression not only

had a higher percentage of improved transformants, but h-

glucosidase levels were considerably higher than those for

the other combinations. Overall, the screening data suggest

that BiP and PDI expression characteristics (constitutive vs.

Table II. BiP, PDI, and h-glucosidase levels from improved system transformants.

Relative

increase

System name Strain # SD/SG

h-glucosidase

(AM pNPG/min�OD) BiP (AU) PDI (AU)

Approx.

h-glucosidase BiP PDI

GAL-KAR2 + GAL-PDI 16 SD 30.3 F 4.5 26.9 F 4.0 1.3 0.9

SG 2.28 F 0.30 67.3 F 4.8 61.8 F 4.6 2.4 2.8 2.1

62 SD 36.2 F 16.9 41.1 F 13.8 1.5 1.4

SG 1.52 F 0.14 149 F 0 73.1 F 10.6 1.6 6.2 2.5

71 SD 22.3 F 1.2 41.5 F 2.6 0.9 1.4

SG 0.92 F 0.16 164 F 12 56.8 F 13.3 1.0 6.8 2.0

84 SD 27.9 F 3.4 27.8 F 8.4 1.2 1.0

SG 2.11 F 0.11 77.0 F 4.1 28.4 F 1.1 2.2 3.2 1.0

GAL-KAR2 + GAPDH-PDI 40 SD 20.7 F 4.7 94.8 F 5.2 0.9 3.3

SG 2.16 F 0.00 86.6 F 2.2 95.8 F 3.8 2.3 3.6 3.4

67 SD 25.5 F 2.1 26.4 F 0.3 1.1 0.9

SG 1.48 F 0.13 120 F 1 35.6 F 1.3 1.5 5.0 1.2

72 SD 27.1 F 1.9 30.1 F 7.8 1.1 1.1

SG 1.19 F 0.12 44.4 F 5.3 31.4 F 1.2 1.2 1.8 1.1

76 SD 20.4 F 1.2 35.2 F 3.9 0.8 1.2

SG 1.68 F 0.24 63.3 F 2.9 31.2 F 1.1 1.8 2.6 1.1

80 SD 15.2 F 0.7 21.0 F 1.5 0.6 0.7

SG 1.17 F 0.29 123 F 11 41.2 F 1.1 1.2 5.1 1.4

GAPDH-KAR2 + GAL-PDI 1 SD 83.5 F 4.5 25.7 F 2.9 3.5 0.9

SG 2.18 F 0.26 79.3 F 2.1 56.9 F 3.1 2.3 3.3 2.0

4 SD 36.4 F 1.3 37.7 F 1.2 1.5 1.3

SG 2.54 F 0.12 37.1 F 3.9 114 F 4 2.7 1.5 4.0

9 SD 16.9 F 4.4 31.7 F 4.5 0.7 1.1

SG 2.17 F 0.20 21.6 F 0.5 239 F 10 2.3 0.9 8.4

11 SD 32.2 F 1.1 41.6 F 7.9 1.3 1.5

SG 2.45 F 0.18 31.9 F 3.0 224 F 8 2.6 1.3 7.9

12 SD 58.9 F 7.2 24.3 F 0.8 2.4 0.8

SG 2.65 F 0.35 64.8 F 3.2 114 F 6 2.8 2.7 4.0

15 SD 22.2 F 2.3 14.8 F 6.6 0.9 0.5

SG 2.06 F 0.51 47.0 F 18.6 146 F 58 2.2 2.0 5.1

16 SD 73.6 F 1.0 27.9 F 4.3 3.1 1.0

SG 2.95 F 0.18 69.1 F 0.7 284 F 15 3.1 2.9 9.9

GAPDH-KAR2 + GAPDH-PDI 6 SD 63.3 F 2.0 120 F 11 2.6 4.2

SG 1.93 F 0.24 63.4 F 0.6 153 F 32 2.0 2.6 5.3

7 SD 24.1 F 0.7 235 F 5 1.0 8.2

SG 2.15 F 0.10 32.5 F 0.8 239 F 21 2.2 1.3 8.4

11 SD 29.4 F 6.8 213 F 10 1.2 7.5

SG 2.24 F 0.17 38.2 F 2.6 202 F 9 2.3 1.6 7.1

BJ5464-h SD 21.9 F 2.3 25.8 F 0.9 0.9 0.9

SG 0.96 F 0.04 33.9 F 4.4 23.7 F 3.1 1 1.4 0.8

BJ5464-C SD 24.1 F 0.3 28.9 F 2.3 1.0 1.0

SG 0.03 F 0.03 17.1 F 0.9 9.0 F 3.3 0.7 0.3

A subset of improved strains from Figure 1 were grown and expressed in duplicate as in Table I. Cell samples were taken in SD and SG media and

analyzed for BiP and PDI levels by Western blot analysis. SG culture supernatants were assayed for h-glucosidase activity as before (reported in AM pNPG/

min/OD). BiP and PDI levels were measured by densitometric analysis of Western blots and reported in arbitrary units. Transformant h-glucosidase levels

were normalized relative to the control (BJ5464+pRS314-h), and BiP and PDI levels were normalized to the overall control (BJ5464+pRS314). Boldface

entries highlight those samples that were used for expression vs. time studies (Fig. 3). Reported errors represent the standard deviation between duplicate

expression experiments. Errors in "approximate relative increases" were typically F10%.


inducible) play an important role in improved h-glucosi-

dase secretion.

Investigation of Improved Strains

To examine these improvements in more detail, a subset of

improved transformants from each system was re-expressed

in 5-mL cultures. In all, four GAL-KAR2+GAL-PDI+h,

five GAL-KAR2+GAPDH-PDI+h, seven GAPDH-KAR2+

GAL-PDI+h, and three GAPDH-KAR2+GAPDH-PDI+htransformants were investigated further. Transformants

GAL-KAR2+GAPDH-PDI+h #80 and GAL-KAR2+GAL-

PDI+h #71 had secretion levels close to that of the BJ5464-hcontrol, and were used as a type of ‘‘negative control.’’

The 19 transformants and 2 controls (BJ5464-C and

BJ5464-h) were grown and expressed as before. Cell

samples were taken in both SD and SG minimal media and

intracellular BiP and PDI levels were determined using

Western blot analysis. Cell culture supernatants were

isolated from SG media cultures and assayed for h-

glucosidase activity. BiP, PDI, and h-glucosidase values

for all samples in both SD and SG media are summarized

in Table II.

Unlike the case of multicopy h-glucosidase expression

(JSIY017 cells in Table I), BJ5464-h BiP and PDI levels

appeared similar to the BJ5464-C control. BiP and PDI

levels did not change significantly with the single gene

copy of h-glucosidase (BJ5464-h), whereas both proteins

increased approximately fourfold in JSIY017-expressing

cells. Therefore, it appears that, although both cells have

difficulty in properly exporting h-glucosidase (Smith and

Robinson, 2002), the added h-glucosidase gene copies and

increased unfolded protein in the ER of JSIY017 appeared

to cause a considerably higher stress on these cells.

A plot of the relative BiP and PDI levels derived from

SG cultures (Table II) shows that a wide range of both PDI

and BiP levels enabled secretion improvements (Fig. 2A).

However, transformants tended to have high BiP and low

PDI levels, high PDI and low BiP levels, or moderate levels

of each. In no case was a transformant isolated with high

levels of both BiP and PDI. This may indicate a true trade-

off in expression, or may be an artifact of the limited

sampling. Also, contrary to the observation from the overall

screen (Fig. 1), inducible vs. constitutive expression did not

appear to play as large a role as simply the total PDI or BiP

present in the cell.

Significant trends were found by plotting the relative

increase in h-glucosidase levels vs. that of either PDI or

BiP. Increasing intracellular BiP levels decreased improve-

ments in h-glucosidase secretion (Fig. 2C). This was

especially evident for the ‘‘negative controls’’ GAL-

KAR2+GAPDH-PDI+h #80 and GAL-KAR2+GAL-PDI +h#71, which had high levels of BiP, low levels of PDI, and

approximately the same h-glucosidase secretion levels as

BJ5464-h. PDI levels showed an opposite correlation,

where increased PDI levels increased h-glucosidase levels

(Fig. 2B).

The complication in examining the response of h-

glucosidase to PDI and BiP is that BiP and PDI work

synergistically, as observed from single BiP/PDI experi-

ments (Table I). Because BiP and PDI are not independent

of one another, the effects of BiP and PDI seen in Figure 2B

and C cannot be completely decoupled. From Figure 2A we

see that low BiP levels were typically accompanied by high

PDI and vice versa. This may explain why the data in

Figure 2B and C were more scattered at low BiP/PDI con-

centrations and then became less varied at high levels. The

dependence of h-glucosidase at low levels of BiP or PDI

actually includes a significant contribution by the other pro-

tein. For example, in Figure 2B, samples with low PDI and

h-glucosidase levels below the trend line tended to have

high levels of BiP.

Transient Detection of BiP, PDI, and B-Glucosidase

The trends seen in Figure 2 resulted from static timepoint

experiments. To more completely characterize the effects of

BiP and PDI on h-glucosidase secretion to determine whe-

ther we could clarify the role of these proteins, we followed

BiP, PDI, and intracellular h-glucosidase levels over time.

Cultures of GAPDH-KAR2+GAL-PDI+h #16, GAPDH-

KAR2+GAPDH-PDI+h #6, GAL-KAR2+GAPDH-PDI+h#40, and BJ5464-h were sampled at multiple timepoints

Figure 2. Graphical presentation of Table II data. Relative levels of BiP,

PDI, and h-glucosidase derived from SG media cultures were plotted

against each other for system GAL-KAR2+GAL-PDI+h (.), GAL-

KAR2+GAPDH-PDI+h (o), GAPDH-KAR2+GAL-PDI+h (n), and

GAPDH-KAR2+GAPDH-PDI+h (5). Plots show the relative levels of

PDI vs. BiP (A), h-glucosidase vs. PDI (B), and h-glucosidase vs. BiP (C)

over the controls (BJ5464-h for h-glucosidase levels and BJ5464-C for

BiP and PDI levels). Lines shown for (B) and (C) indicate the linear least-

squares fit of the data. R2 values for both plots were approximately 0.4. (D)

BiP and PDI Western blot images for represented transformants in both SD

and SG media. Errors in relative BiP and PDI levels, as determined by the

standard deviation of duplicate samples quantified by Western blot

analysis, are shown for (A).


postinduction (SG media), and analyzed by Western blots for

BiP, PDI, and h-glucosidase levels (Fig. 3). These four

transformants were chosen for their range of BiP, PDI, and

secreted h-glucosidase levels as well as their variation in

expression modes. The 0- and 48-h timepoints were in good

agreement with the data from Table II. Stationary phase for

all four cultures was reached at approximately 40 to 48 h.

As presented in Figure 3, the onset of h-glucosidase

secretion and the rate of accumulation did not appear to be

affected by the levels or expression modes (constitutive or

inducible) of BiP and PDI. h-Glucosidase levels quickly

rose to approximately maximal levels within 16 to 20 h

postinduction. BiP and PDI levels also reached their highest

levels within this time frame. Overall, the behavior of BiP

and PDI was as expected for promoter regulation.

However, an interesting observation was made from

examination of BJ5464-h. In BJ5464-h control cells

(Fig.3D), BiP and h-glucosidase levels rose throughout the

course of the experiment. PDI levels for this strain showed a

small initial induction (f60%) followed by a return to

approximately initial conditions. The induction and repres-

sion of PDI seen in BJ5464-h may indicate PDI induction in

an unfolded protein response of the cell. A smaller induction

(f20%) was observed in GAL-KAR2+GAPDH-PDI+h #40.

This effect may not have been seen with the other two cell

strains because PDI levels were already elevated to high

levels (9.9- and 5.3-fold vs. 3.4-fold relative to BJ5464-C for

GAL-KAR2+GAPDH-PDI+h #40; Table II).

A change in the ER bottleneck might be observed by

comparing the levels of intracellular h-glucosidase. A

comparison of the intracellular h-glucosidase in all four

transformants is shown in Figure 4. The best secretion

transformant, GAPDH-KAR2+GAL-PDI+h #16, showed an

earlier decrease in intracellular h-glucosidase than the other

three after approximately 8 h, and reached the lowest final

levels of the four transformants. Intracellular h-glucosidase

levels for GAPDH-KAR2+GAPDH-PDI+h #6 and GAL-

KAR2+GAPDH-PDI+h #40 were approximately equal to

that of BJ5464-h until after approximately 16 h, and

reached final levels much lower than that of BJ5464-h.

Intracellular levels were inversely related to the secreted

levels of h-glucosidase for these transformants (Table II).

Because BiP levels were relatively equal among the trans-

formants, the data suggest that PDI acts to lower intra-

cellular h-glucosidase levels that, in turn, leads to increased

secreted h-glucosidase.

DISCUSSION

Single BiP/PDI Gene Expression

We previously reported the existence of an intracellular ER

bottleneck in our high h-glucosidase gene copy S. cerevisiae

transformant, JSIY017 (Smith and Robinson, 2002). There

are numerous instances in the literature wherein over-

expression of ER chaperones and foldases has been used to

increase secretion yields. Therefore, we investigated the

effect of single gene doses of the ER-resident proteins BiP

and PDI on h-glucosidase secretion in JSIY017.

Overexpression of both BiP and PDI synergistically

improved h-glucosidase secretion. Interestingly, this type

of result has been seen with recombinant single-chain

antibody secretion in S. cerevisiae, wherein overexpression

of BiP and PDI increased scFv secretion levels higher than

the additive effects of BiP and PDI alone (Shusta et al.,

Figure 3. Intracellular BiP, PDI, and h-glucosidase levels over time.

GAPDH-KAR2+GAL-PDI+h #16 (A), GAPDH-KAR2+GAPDH-PDI+h #6

(B), GAL-KAR2+GAPDH-PDI+h #40 (C), and BJ5464-h (D) were grown

to saturation in SD media. Also, 50 OD-mL equivalents of cells were

isolated and resuspended in 50 mL SG media (t = 0). Cell samples were

taken at various timepoints postinduction and analyzed by Western blot for

h-glucosidase (x), BiP (5), and PDI (o). All species were normalized by

the highest signal for that species.

Figure 4. Intracellular h-glucosidase levels over time. Non-normalized

h-glucosidase levels for GAPDH-KAR2+GAL-PDI+h #16 (n), GAPDH-

KAR2+GAPDH-PDI+h #6 (5), GAL-KAR2+GAPDH-PDI+h #40 (o), and

BJ5464-h (E) are plotted for direct comparison. Intracellular h-

glucosidase levels were measured by Western blot analysis and reported

in arbitrary units.


1998). This effect was also seen in the in vitro refolding of

reduced Fab fragments in the presence of BiP and PDI

(Mayer et al., 2000). Buchner and colleagues suggested that

BiP held open the folding intermediate, which gave PDI

access to the disulfide bonds. However, because P. furiosus

h-glucosidase has no disulfide bonds and only one cysteine

per monomer, PDI’s role as a foldase would not explain its

effect here.

Overexpression of BiP from a single-copy plasmid

pGalKar2-LEU in JSIY017 did not change BiP protein

levels. Perhaps the cell autoregulates BiP levels though

transcriptional or translational control in this case. Interest-

ingly, PDI levels do not respond in a similar manner.

Overexpression of PDI leads to a twofold increase over

JSIY017 control cells from SG media when PDI is

overexpressed by itself or in concert with BiP. Both PDI

and BiP are a part of the unfolded protein response (UPR)

(Chapman et al., 1998; Mori et al., 1998); however, our data

may suggest that BiP and PDI protein levels are controlled in

different ways.

Multicopy BiP/PDI Optimization

Due to the possible integration and selection difficulties of

using pITy-based h-glucosidase and BiP/PDI plasmids, we

employed the pITy-based BiP/PDI plasmids to improve

secretion of h-glucosidase from wild-type BJ5464 trans-

formed with the CEN-ARS zpRS314-h plasmid (Smith and

Robinson, 2002). Therefore, the utility of the varying BiP

and PDI levels was determined so that in the future this

knowledge culd be applied to high h-glucosidase expression

systems or expression systems with other hetero-

logous proteins.

In this study we optimized h-glucosidase secretion in

BJ5464 cells transformed with pRS314-h by varying BiP

and PDI levels through random chromosomal BiP/PDI gene

integration and then screening for higher h-glucosidase

secretion levels (Fig. 1). A subset of improved trans-

formants was then examined for intracellular h-glucosidase

as well as BiP and PDI levels (Fig. 2). Two general trends

emerged: increasing BiP levels decreased h-glucosidase

secretion, whereas increasing PDI levels increased h-

glucosidase secretion.

Increasing BiP levels may result in decreased h-

glucosidase secretion due to prolonged interaction with

immature h-glucosidase. Secretory proteins tend to have a

unique, individual average residence time in the ER, due in

part to the folding-mediated effects of ER-resident chaper-

ones and foldases (Pelham, 1989; Rowling and Freedman,

1993). Typically, the shorter the residence time in the ER,

the faster the proteins are secreted. On the other hand, if

chaperone levels are increased, ER residence times may

increase due to prolonged interaction with ER-resident

proteins and lower overall secretion yields. As an example,

elevated levels of Grp94p, an ER chaperone, were found to

increase the residence times for thyroglobulin in Chinese

hamster ovary cells (Muresan and Arvan, 1997).

However, from single BiP/PDI gene overexpression

results, we found that BiP and PDI together improved h-

glucosidase secretion. Therefore, although a positive role

for BiP at increased levels was not seen (Fig. 2), data for

samples GAPDH-KAR2+GAL-PDI+h #12 and #16 may

indicate that increased levels of BiP were necessary, albeit

at moderate levels.

In contrast to BIP effects, increased PDI levels increased

h-glucosidase levels (Fig. 2B). Figure 4 suggests that

increased PDI functions to lower intracellular h-glucosi-

dase, which leads to elevated secreted h-glucosidase yields.

However, the role of PDI in h-glucosidase folding and

secretion is less clear, because h-glucosidase lacks

disulfide bonds.

We postulate two possible modes of action for PDI.

Although the mechanism of action is not well understood, at

stoichiometric levels PDI can act as a chaperone (Dai and

Wang, 1997; Klappa et al., 1998; LaMantia and Lennarz,

1993). It is possible PDI may act as a chaperone for h-

glucosidase in a way that BiP cannot, a role that does not

become saturating, even at high PDI levels. Alternatively,

PDI may be forming mixed, transient disulfide bonds with

the h-glucosidase, helping to segregate its folding environ-

ment or limiting its conformational explorations in the

process of proper folding.

Thaumatin secretion was improved by increasing intra-

cellular PDI levels two- to threefold; however, according to

Moralejo et al. (2001), increasing PDI above this level

results in decreased overall yields. They reasoned that the

high levels of PDI may unfavorably alter the folding

equilibrium, leading to nonsecretable forms of thaumatin.

At high levels, PDI has been found to act as an anti-

chaperone and aggregate immature proteins regardless of

their disulfide character (Quan et al., 1995). Therefore, one

could imagine that an optimum level of PDI might be found

if it were acting as a chaperone. However, for h-glucosidase,

only a solely beneficial role for PDI was observed.

In preliminary efforts to examine the possible foldase role

of PDI in vivo, DTT was added to the culture media in an

attempt to inhibit PDI’s foldase ability. We grew four

selected transformants in SG media containing 1 mM and

5 mM DTT. Cell yields were significantly hampered in the

presence of DTT, reaching approximately 60% and 10% of

final ODs, respectively, for the 1 mM and 5 mM DTT cases,

compared with expression in the absence of DTT (data not

shown). On a per-cell basis, h-glucosidase secretion was

approximately equal for all samples in the absence or

presence of DTT (data not shown). Therefore, these results

suggest that PDI does not form mixed disulfides, but rather

acts primarily as a chaperone. However, DTT is used widely

to invoke the cellular unfolded protein response (Gasch et al.,

2000), which causes a shift in the expression of many genes,

including the ER-resident proteins BiP and PDI. As a result,

direct effects of DTT are not completely conclusive.

Additional studies are being conducted to test the effect

of site-directed mutagenesis of the sole cysteine in h-

glucosidase on its secretion and the role of PDI. We also


plan to study PDI interactions with h-glucosidase in in vitro

refolding studies. By refolding h-glucosidase in the

presence of PDI mutants, which possess only a chaperone

(Wang, 2002) or a foldase ability (Dai and Wang, 1997),

the role of PDI could possibly be deduced.

Impact on Heterologous Protein Secretion

Often, random screens of different cell strains are

conducted to find an optimal strain for production. Here

we attempted a more rational cellular engineering approach

based on the determination of an ER bottleneck. Overall,

we have successfully created a system allowing for

screening of a range of BiP and PDI levels to improve

heterologous protein secretion. Relative to the wild-type

strain (containing one h-glucosidase gene), secretion was

improved approximately threefold in the best strain, and for

that strain PDI and BiP levels were approximately ten- and

threefold, respectively, higher than with the wild-type cells.

The scope of this approach is significantly broader than

that of previous studies of chaperone overexpression effects.

It allows for not only the selection of optimal PDI and BiP

levels, but also the identification of whether these proteins

aid or inhibit heterologous protein secretion. BiP and PDI

screening could easily be supplemented to include other

chaperones. This approach could be particularly useful in the

biotechnology industry, wherein many of the therapeutic

proteins are complex and require the assistance of chaper-

ones and foldases.

The trends seen here with h-glucosidase are likely not

directly translatable to other proteins, as different proteins

have demonstrated different behaviors in response to mole-

cular chaperones. In addition, the chaperone/foldase needs

of a single protein may change depending on environmental

changes. For example, h-glucosidase requirements at 37jC

expression are distinct from those at 30jC (unpublished

data); high PDI levels appear to lower h-glucosidase se-

cretion at 37jC.

CONCLUSIONS

We previously reported a high h-glucosidase gene copy

Saccharomyces cerevisiae transformant that could secretef12 mg/L, but also retained a large intracellular bottleneck

in the ER. In an effort to relieve this bottleneck, an

additional BiP and PDI gene was introduced into these cells,

either alone or in combination. A synergistic effect was seen

between PDI and BiP, resulting in f60% improved

secretion levels. Due to this modest increase, we attempted

to optimize the effects of BiP and PDI on h-glucosidase

secretion by randomly varying integrated BiP/PDI gene

copynumbers. Transformants were screened for improvedh-

glucosidase secretion and a subset of improved strains were

re-examined for h-glucosidase secretion and intracellular

BiP and PDI levels. Two general trends emerged: increasing

BiP levels decreased h-glucosidase secretion, whereas

increasing PDI increased h-glucosidase secretion. However,

these relationships are not independent of each other and

deviations from the overall trend are especially evident at

low BiP and PDI levels. Increased PDI levels decreased the

levels of intracellularly retained h-glucosidase. Although

preliminary results indicate that PDI acts as a unique

chaperone for h-glucosidase, PDI may possibly be forming

mixed disulfides with h-glucosidase during the folding

process. Overall, we have successfully created a screening

system allowing for qualitative understanding of multiple

chaperone/foldase effects on a heterologous protein.

The authors thank Jeff Brodsky (University of Pittsburgh), K. Dane

Wittrup (MIT), and Bob Kelly (North Carolina State University) for

their generous gifts of the BiP antibody, pITy plasmid, and h-

glucosidase gene, respectively.

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