Optimization of the wheat puroindoline-a production in Pichia pastoris

Optimization of the wheat puroindoline-a production inPichia pastoris

N. Issaly, O. Solsona, P. Joudrier1, M.F. Gautier1, G. Moulin and H. BozeUFR Microbiologie Industrielle et de GeÂneÂtique des Microorganismes, ENSA.M-INRA, Montpellier and 1UniteÂ

de Biochimie et Biologie MoleÂculaire des CeÂreÂales, INRA Montpellier, France

494/08/00: received 2 August 2000, revised 9 November 2000 and accepted 14 November 2000

N. ISSALY, O. SOLSONA, P. JOUDRIER, M.F . GAUTIER, G. MOULIN AND H. BOZE. 2001.

Aims: A recombinant puroindoline-a (rPIN-a) was produced using the methylotrophic yeast

Pichia pastoris.

Methods and Results: In fed-batch culture, the production of rPIN-a decreased after 24 h of

methanol induction. Most of the rPIN-a was not soluble in the culture medium remaining

bound to the cell walls. Soluble and membrane-bound rPIN-a were quanti®ed by ELISA after

Triton X-114 phase partitioning. In order to improve the production of rPIN-a, the in¯uence

of pH, speci®c growth rate and the addition of TX-114 was tested on two independent

continuous cultures. The production of rPIN-a was improved when continuous culture was

carried out at 29°C under acid conditions (pH 5) with a low dilution rate (D � 0á025 h)1). The

addition of 0á01% TX-114 to the medium inverted the ratio between the secreted and the

membrane-bound rPIN-a.

Conclusions: When a continuous culture was carried out under optimized conditions, the

rPIN-a production yield was increased 10-fold to 14 mg l)1 and 80% of the rPIN-a was

soluble.

Signi®cance and Impact of the Study: This study would be helpful to optimize the

expression of other membrane-bound proteins in P. pastoris.

INTRODUCTION

Puroindolines are basic cystine-rich proteins of about

13 kDa that have been isolated from wheat endosperm by

the Triton X-114 phase partitioning method (Blochet et al.1993). They belong to cystine-rich proteins of wheat seed as

purothionins, lipid transfer proteins and CM proteins. Two

cDNA clones encoding puroindoline-a (PIN-a) and puro-

indoline-b (PIN-b) have been characterized. They are 55%

similar and contain 10 cystine residues involved in ®ve

disulphide bonds which maintain their folded conforma-

tions. Furthermore, these proteins exhibit a unique trypto-

phan-rich domain which is partially truncated in PIN-b

(Gautier et al. 1994). Puroindolines are related to basic

friabilins (Greenblatt et al. 1994) and starch granule proteins

(Rahman et al. 1994). The presence of these proteins has

been correlated to wheat kernel softness (Jolly et al. 1993;

Sourdille et al. 1996). In wheat endosperm, puroindolines

are localized in the starchy endosperm and in the aleurone

layer (Dubreil et al. 1998). Phase partitioning in a non-ionic

detergent, TX-114, a method that usually speci®cally

extracts membrane proteins (Bordier 1981), suggests that

puroindolines might interact with polar lipids. Whereas

PIN-b interacts only with negative phospholipids, PIN-a is

capable of binding tightly to both wheat phospholipids and

glycolipids (Dubreil et al. 1997). This tight binding of

puroindolines to polar lipids, which has also been observed

with purothionins, could be responsible for their membra-

notoxic effects which play a role in the defence mechanism

against pathogens (Stuart and Harris 1942; Le GuerneveÂ

et al. 1998; Mourgues et al. 1998). Puroindoline-a has

intrinsically good foaming properties and prevents the

lipid-induced destabilization of protein beer foam (Clark

et al. 1994), suggesting that lipid binding may also occur at

the air±water interface. Furthermore, the unique surface

properties of puroindolines make them attractive in bread-

making technology (Dubreil et al. 1998). Le Bihan et al.(1996) observed, by infrared and Raman spectroscopy, that

Correspondence to: H. Boze, UFR Microbiologie Industrielle et de GeÂneÂtique

des Microorganismes, Institut National de la Recherche Agronomique, 2 place

Viala, 34060 Montpellier cedex 01, France (e-mail: [email protected]).

ã 2001 The Society for Applied Microbiology

Journal of Applied Microbiology 2001, 90, 397±406

under acidic and high ionic strength conditions PIN-a tends

to form aggregates.

Considering puroindolines to be important proteins in

future breeding programs and regarding their numerous

technological applications we produced recombinant PIN-a

(rPIN-a) in order to understand its structure±function

relationships. The methylotrophic yeast Pichia pastoris has

the ability to utilize methanol as a sole source of carbon and

energy and has been used extensively as a host system for

the expression of foreign genes (Sreekrishna et al. 1988;

Cregg et al. 1993). The highly ef®cient P. pastoris alcohol

oxidase 1 gene (AOX1) promoter was used for the high-level

expression of foreign genes. This expression system is

particularly valuable for its ability to secrete heterologous

proteins with high ef®ciency. In this paper we report the

production of rPIN-a in a 1á5-l working volume fermenter.

Because of its lipid-binding properties, a large proportion

of rPIN-a is not soluble in the culture medium. To test

whether rPIN-a remains bound to the cell membrane, we

investigated a derivated TX-114 phase partitioning protocol

to make rPIN-a soluble. Firstly, we tried to optimize rPIN-a

production in both fed-batch and continuous culture. The

in¯uence of parameters such as pH, temperature and growth

rate during continuous fermentation was studied, as well as

the addition of Triton X-114 and casamino acids to the

culture medium. The production of rPIN-a was then carried

out using these optimized parameters.

MATERIALS AND METHODS

Yeast and Escherichia coli strains

Escherichia coli strain JM109 was used for all plasmid

constructions. Pichia pastoris strain GS115 (his4) was

obtained from Invitrogen BV (Leek, The Netherlands)

and vector pYAM7SP8 from Dr Laroche (Leuven Catholic

University, Belgium).

Construction of pYAMPIN-a expression vectorand Pichia pastoris transformation

The PIN-a coding sequence from the pTa-31 clone (Gautier

et al. 1994) was ampli®ed by polymerase chain reaction

(PCR) creating a HindII restriction site at the 5¢ end

and an XhoI restriction site at the 3¢ end using the

AP1 (5¢-AGTGTCGACGTTGCTGGCGG-3¢) and AM1

(5¢-TATAGACACCTCGAGCAGGC-3¢) primers. The

HindII-XhoI sequence was then inserted in the pYAM7SP8

vector (Laroche et al. 1994) after cleavage by StuI-XhoI

restriction enzymes. Pichia pastoris strain GS115 (his4) was

transformed with either 20 lg NotI-linearized pYAMPIN-a

or SalI-linearized pYAMPIN-a using the spheroplast

method (Invitrogen) and plated on RDB plates [1 mol l)1

sorbitol, 1% dextrose, 1á34% yeast nitrogen base with

ammonium sulphate and without amino acids, 4 ´ 10)5 %

biotin, 5 ´ 10)3 % amino acids (LL-glutamic acid, LL-lysine,

LL-methionine, LL-leucine and LL-isoleucine) and 2% agar].

His+ transformants were screened for the Mut+/Muts

phenotype by spotting the His+ transformants on minimal

dextrose (MD) medium agar plates (1á34% yeast nitrogen

base with ammonium sulphate and without amino acids,

4 ´ 10)5% biotin, 1% dextrose and 1á5% agar) and minimal

methanol (MM) medium agar plates (as for MD but with

0á5% methanol).

Selection and screening of transformants

The rPIN-a production of different transformed yeasts was

tested using the following method. Each clone was inocu-

lated in 10 ml YMPG medium (0á3% yeast extract, 0á3%

malt extract, 0á5% bactopeptone and 1% glycerol) and

grown overnight in shake ¯asks at 28 °C (80 oscillations

min)1, amplitude 7 cm). After a 10-min centrifugation at

1000 g, the pellet of each clone was diluted with ultra-pure

H2O so that cultures had the same A600 value, approximately

equal to 1. Serial dilutions to 10)5 with ultra-pure water

were carried out and 5 ll of each dilution were spotted onto

MM agar plates. After 24, 48 and 72 h incubation at 28 °C,

the colony-forming units were blotted onto Hybond C extra

(Amersham France S.A., Courtaboeuf, France). Membranes

were soaked for 30 min at room temperature in TBS buffer

(20 mmol l)1 Tris-HCl, pH 7á5 and 0á15 mol l)1 NaCl)

containing 0á1% (v/v) Tween 20 and then incubated for 1 h

in the same buffer containing 1% (w/v) bovine serum

albumin (BSA) and a 1:1000 dilution of an antiserum raised

against the wheat puri®ed PIN-a. Membranes were washed

three times in TBS buffer (10 min each) and primary

antibodies revealed by incubation for 1 h with a 1:10 000

dilution of phosphatase alkaline-labelled goat antirabbit IgG

(Promega, Sigma Chemical Co., St. Louis, MO, USA) in

BSA-Tween TBS buffer. Detection was carried out using an

enzymatic colourimetric assay with a mix of 5-bromo-4-

chloro-3-indolyl phosphate and nitroblue tetrazolium salt in

a 0á1 mol l)1 Tris-HCl, pH 9á5, 0á1 mol l)1 NaCl buffer.

Culture conditions

Cultures were carried out largely as described by Klein et al.(1998). All cultures in shake ¯asks were performed at 29 °C

and under aerobic conditions (80 oscillations min)1, ampli-

tude 7 cm). A 10-ml seed culture of GS115 pYAMPIN-a in

YMPG medium was grown for 24 h and then used to

inoculate 20 ml of a synthetic medium [4% glycerol,

1 ´ PTM1 trace elements, 10 ´ FM21 basal salt and

120 lg l)1 d-biotin (Sreekrishna et al. 1989), buffered at

pH 5á4 with a nitrogen source buffer (0á75% tartric acid,

398 N. ISSALY ET AL .

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 397±406

3á58% Na2HPO4, 12 H2O (dodeca hydrated), 0á2%

SO4(NH4)2 and 0á6% H2PO4NH4)]. This culture was

incubated for 24 h and then transferred into 200 ml of the

same synthetic medium. After 24 h of culture, 200 ml

inoculum were transferred into a 3-l fermenter vessel

(Applikon, Schiedam, The Netherlands) containing 1á5 l

synthetic medium (4% glycerol, 1 ´ PTM1 trace elements,

10 ´ FM21 basal salt and 80 lg l)1 d-biotin). The pH was

regulated with 15% (v/v) ammonium hydroxide, which was

also used as a nitrogen source, and 2 NN H2SO4. The fed-

batch feeding rate was increased from 1á2 to 7á2 ml h)1

during 144 h of induction with a synthetic medium

(682 g l)1 methanol, 1á2 ´ PTM1 and 2á3 mg l)1 d-biotin).

The synthetic medium used for continuous cultures was

composed of 226 g l)1 methanol, 17á5 ´ FM21, 7á5 ´ TM1

and 1á5 mg l)1 d-biotin. During the batch and methanol

induction phases, the pH and temperature were controlled

and dissolved oxygen maintained above 30%.

Determination of cell density

Cell growth was monitored by measuring the absorbance at

600 nm using a spectrometer (DU7; Beckman Instruments

Inc., Fullerton, CA, USA). One A600 unit corresponds to

0á236 � 0á001 g dry weight l)1 on glycerol growth in batch

culture and 0á221 � 0á085 g dry weight l)1 on methanol

growth in fed-batch or continuous cultures.

Analyses of substrates and metabolites

The separation of the cells and supernatant ¯uid was

performed by automatic sterile ®ltration with a Water

Filter/Acquisition module. Glycerol, methanol and formal-

dehyde were separated by a Watersä Fast Fruit Juice

column (Waters Corporation, Milford, MA, USA) in ion

exclusion mode. The mobile phase was 3 mmol l)1 ortho-

phosphoric acid and the ¯ow rate was 1 ml min)1. The

detection was performed by refractometry (410; Waters).

The lower detection limit of the high performance liquid

chromatography was 0á1 g glycerol l)1, 0á1 g methanol l)1

and 0á2 g formaldehyde l)1.

Extraction and solubilization of recombinantpuroindoline-a

The TX-114 phase partitioning was performed on 50 ml

culture during the fed batch phase. Soluble rPIN-a was

extracted for 2 h at 4 °C by gently stirring the sample. After

a ®rst 15-min centrifugation at 8000 g at 4 °C the pellet was

stirred again with 50 ml TX-114 buffer [0á1 mol l)1 Tris-

HCl, pH 7á8, 5 mmol l)1 EDTA, 0á1 mol l)1 KCl and 4%

(v/v) Triton X 114]. This operation was repeated twice on

the pellet. The supernatant ¯uid containing rPIN-a was

heated to 38 °C for 1 h and centrifuged for 15 min at

8000 g at 4 °C. The upper detergent-poor phase was

discarded and rPIN-a present in the rich detergent phase

was precipitated overnight with two volumes of 80% (v/v)

acetone at )20°C. After a 15-min centrifugation at 8000 gand 4 °C, the protein pellet was washed three times with

80% acetone, dried and concentrated 16-fold in ultra-pure

water.

Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis and Western blot analysis

Both the culture supernatant ¯uids and TX-114-rich phase

were analysed on denaturing sodium dodecyl sulphate-

polyacrylamide (18%, w/v) gel electrophoresis under redu-

cing conditions (Laemmli 1970) followed by staining with

Coomassie brillant blue R or immunoblotting. Molecular

weight markers were obtained from GibcoBRL (Life

Technologies, USA) (2850±43 000 molecular range) and

PIN-a isolated from wheat was used as control. For Western

blot analysis, proteins were blotted onto Hybond C extra

(Amersham France S. A., Courtaboeuf, France) with a semi-

dry electro transfer Biometra (Biometra Rudolf-Wissell,

GoÈttingen, Germany) apparatus. Membranes were soaked

for 30 min in TBS buffer (20 mmol l)1 Tris-HCl, pH 7á5,

0á15 mol l)1 NaCl) containing 1% (w/v) BSA and 0á1% (v/

v) Tween 20. The same procedure as described for screening

of the transformant was used.

Immunochemical determination of recombinantpuroindoline-a content

The enzyme linked immunosorbent assay (ELISA) was

performed by immobilizing direct supernatant ¯uid or

TX-114 extracts on 96-well microtiter plates. Each well

was coated overnight at 4 °C with 100 ll of the wheat

puri®ed PIN-a in the 2á5±80 ng ml)1 range or with

suitable sample dilutions in a phosphate-buffered saline

(PBS) buffer, pH 6á9 (8 mmol l)1 Na2HPO4, 1á5 mmol l)1

KH2PO4, 0á14 mol l)1 NaCl and 2á7 mmol l)1 KCl)

containing 1% (w/v) BSA. The plate was washed three

times with the PBS buffer containing 0á05% (v/v) Tween

20. Phosphate-buffered saline-BSA (200 ll) was added

and incubated for 1 h at 37 °C. The plate was washed a

further three times and 100 ll of the polyclonal PIN-a

antibody (dilution 1:2000) added and incubated for 1 h at

37 °C. After three washes, primary antibodies were

revealed by the antirabbit IgG biotin (dilution 1:1000)

conjugated with streptavidin-biotinylated horseradish per-

oxidase complex (dilution 1:1000) and incubated for 1 h at

37 °C. The wells were washed a ®nal three times and

100 ll of TMB peroxidase substrate (Kirkegaard & Perry

Laboratory, Goithersburg, Maryland, USA) and hydrogen

PUROINDOLINE-A PRODUCTION BY PICHIA PASTORIS 399


peroxide solution (H2O2) added. The plate was incubated

at room temperature and the enzyme activity stopped by

the addition of 100 ll 1 mol l)1 H3PO4. The absorbance

measurements were performed at 450 nm.

Subcellular fractionation

Yeast cells were harvested by centrifugation at 8000 g for

10 min at 4 °C and washed twice with a equal volume of the

TBS buffer used in the ELISA procedure. Glass beads (8 g

Glasperlen beads, 0á45±0á5 mm, Braun Melsengen, Postfach,

Germany) were added to 10 ml culture and yeasts were

broken for 2 min using a CO2-cooled Braun MSK bead

grinder apparatus (Braun Melsengen, Postfach, Germany).

The homogenate was centrifuged for 20 min at 3000 g at

4 °C and the protein content measured with the biuret

procedure (Stickland 1951) and the ELISA procedure in

both soluble protein fractions within the supernatant ¯uid

and insoluble protein fractions within the pellet.

RESULTS

Construction of the expression vectorand selection of a productive clone

The PIN-a coding sequence was ampli®ed by PCR from the

cDNA clone pTa31 (Gautier et al. 1994) creating a HindII

restriction site at the 5¢ end and a XhoI restriction site at the

3¢ end (Fig. 1). The PCR products were ®rst inserted into

pGEM-T to give rise to the pGEM-T PIN-a and then the

sorted HindII/XhoI fragment was cloned into the StuI-

XhoI-restricted shuttle vector pYAM7SP8 (Laroche et al.1994). The PIN-a encoding sequence was expressed under

the control of the methanol-inducible alcohol oxidase

promoter using the P. pastoris acid phosphatase secretion

signal fused to a synthetic 19 amino acid pro-sequence

ending with a Lys-Arg processing site for the KEX2

protease. After transformation of P. pastoris, 45 His+

transformants were checked for methanol utilization by

replica plating on MM and MD plates in order to

demonstrate phenotypically if the AOX1 gene remained

intact. Each clone at the same cell density was then spotted

onto MM plates for immunodetection. Among the 28 clones

recognized by the PIN-a antibody, two were chosen

according to their higher level of protein synthesis. These

two clones, 44.8.26 and 44.8.36, seemed to be Mut+

according to the MM/MD screening.

Production of recombinant puroindoline-aby fed-batch fermentation

The production of rPIN-a was ®rst tested with clone

44.8.26. Fermentations consisted of both batch and fed-

batch culture procedures. The temperature and pH were

kept, respectively, at 29 °C and 6 during the fermentation. A

®rst fermentation was carried out using glycerol 4% (v/v) in

the initial batch phase. The methanol fed-batch culture was

then initiated when the glycerol was exhausted. In order to

Fig. 1 Construction of the pYAMPIN-a

expression vector. The puroindoline-a

(PIN-a) coding sequence ¯anked by the

HindII and XhoI restriction sites was cloned

into the pGEM-T vector to give rise to

pGEM-TPIN-a. The HindII/XhoI insert was

then inserted into StuI/XhoI-digested

pYAM7SP8 to give rise to pYAMPIN-a.

PCR, Polymerase chain reaction



adapt cells to methanol, an initial period of slow methanol

feeding was ®rst carried out. Methanol feeding was then

regulated in order to keep the speci®c growth rate equal to

0á01 h)1. Under these conditions, no methanol accumulation

was detected during the induction phase. After 143 h of

induction, the ®nal biomass was 73 g l)1 and the biomass

yield on methanol was 0á13 g g)1 (Fig. 2a). Because pre-

liminary dot-blot assays have suggested that rPIN-a behaved

like a membrane-bound protein (data not shown), a

derivated TX-114 phase partitioning was used to follow

the rPIN-a production. The kinetics of rPIN-a production

was followed by analysis of both the culture supernatant

¯uid and the TX-114 lower phase. The production of rPIN-

a reached 1á11 mg l)1 after 22 h of induction, decreased to

0á12 mg l)1 and remained unchanged during the fermenta-

tion process. This decrease corresponds to the growth phase

after the initial adaptation phase. In the same time the rPIN-

a yield decreased from 46á5 to 1á5 lg g)1 and the speci®c

rPIN-a productivity decreased from 0á46 to 0á01 lg g)1 h)1

at the end of the fed-batch culture (Table 1). From the

beginning of induction to 123 h, the proportion of rPIN-a

secreted in the supernatant ¯uid was insigni®cant compared

with the rPIN-a extract from the TX-114 lower phase. An

increase in secreted rPIN-a was observed after 143 h and

36% of rPIN-a was detected in the culture supernatant ¯uid

at the end of the fed-batch culture. The rPIN-a was still

detected after two TX-114 pellet treatments which indicated

the high af®nity of rPIN-a to polar lipids. Three proteins

recognized by the PIN-a antibody were synthesized during

the production phase. According to their molecular mass,

these proteins might correspond to processed (15 kDa) and

unprocessed rPIN-a (about 17 kDa) and the higher one

might correspond to rPIN-a aggregates (Fig. 2b). Most

rPIN-a seemed to be accumulated as the unprocessed form.

In the ®rst few hours following methanol induction, only the

17-kDa rPIN-a was detected. The protein with the expected

Fig. 2 (a) Growth and production of

recombinant puroindoline-a (rPIN-a) during

the fed-batch methanol induction phase with

the 44á8á26 clone. The rPIN-a was estimated

by enzyme-linked immunosorbent assay in

both supernatant ¯uid culture (S1, j) and

after TX-114 phase partitioning (S2, m). d,

Biomass. (b) Western blot analysis of soluble

rPIN-a during fed-batch culture with the

44á8á26 clone. Culture supernatant ¯uid sam-

ples were taken during the methanol induc-

tion phase and analysed by sodium dodecyl

sulphate-polyacrylamide gel electrophoresis

and then by Western blotting with an anti-

serum raised against the wheat-puri®ed PIN-

a. Lanes: 1±6, 8 ll supernatant ¯uid samples

after 45á5, 69á5, 123, 143, 151 and 167 h of

cultivation; 7, 2 ll wheat-puri®ed PIN-a



molecular mass was detected at the beginning of the

induction phase and after 143 h of culture. In order to

determine whether variations in culture conditions have an

effect on the production of rPIN-a and on its solubilization,

the in¯uence of several parameters was tested during

continuous culture.

Optimization of the production of recombinantpuroindoline-a by continuous fermentation

Two continuous cultures were carried out independently with

the 44.8.26 clone. In the ®rst continuous culture, the in¯uence

of physico-chemical parameters and the addition of 5 g l)1

casamino acids on the production of rPIN-a were studied. In

the second continuous culture, the in¯uence of dilution rate

and the addition of 0á01% (v/v) TX-114 were analysed.

In¯uence of the pH, temperature and addition ofcasamino acids. In continuous culture no. 1, the dilution

rate was progressively increased to 0á05 h)1 and kept

constant to give rise to 60 g dry weight l)1. The biomass

yield on methanol was 0á3 g g)1 and the volume change time

was 20 h. Variations of pH, temperature and the addition of

5 g l)1 casamino acids were tested. We considered that a

steady state was reached roughly every 2 d when the

biomass and level of dissolved oxygen were stable. The cell

growth rate did not seem to be affected by the pH variation

from 5 to 7, unlike rPIN-a synthesis which seemed to be

optimal under acid conditions (Table 2). The concentration

of rPIN-a increased from 0á13 mg l)1 at pH 6 to

1á15 mg l)1 after 166 h of cultivation at pH 5. At pH 7,

the concentration of rPIN-a fell to 0á037 mg l)1. The

speci®c rPIN-a productivity was 30 times higher when the

pH was monitored at 5 (q rPIN-a � 0á96 lg g)1 h)1) than

when the pH was regulated at 7 (q rPIN-a � 0á03 lg

g)1 h)1).

In the second steady state, when the temperature was

raised to 37 °C, both the concentration and yield production

of rPIN-a decreased twofold and the solubility of rPIN-a

decreased from 33 to 6% (Table 3).

In order to test whether proteases were responsible for the

decrease in rPIN-a detected in the supernatant ¯uid during

the fed-batch culture, 5 g l)1 casamino acids were added in

the last steady state. The presence of casamino acids did not

have a signi®cant in¯uence on the production of rPIN-a but

seemed to affect the solubility of rPIN-a.

In¯uence of the dilution rate and addition of TX-114.The in¯uence of the dilution rate and addition of TX-114

were analysed in continuous culture no. 2. The continuous

culture was initiated after 120 h with temperature and pH,

respectively, set at 29 °C and 6. The biomass yield on

methanol was 0á3 g g)1. Two dilution rates were ®rstly

tested, 0á03 h)1 in the ®rst steady state and 0á05 h)1 in the

second. The concentration of rPIN-a decreased threefold

from 1á38 to 0á43 mg l)1 when the dilution rate increased,

while the speci®c productivity of rPIN-a decreased from 0á69

to 0á35 lg g)1 h)1 (Table 4). This was in good agreement

with the results obtained during the adaptation phase. The

Table 1 Recombinant puroindoline-a (rPIN-a) concentration

(mg l)1), speci®c productivity (q; lg g)1 h)1) and yield production (Y;

lg g)1) determined by ELISA during the fed-batch culture with the

44.8.26 clone

Time of

methanol

induction (h)

rPIN-a

(mg l)1)

q rPIN-a

(lg g)1 h)1)

Y rPIN-a

(lg g)1)

% rPIN-a

membrane-

bound (S2)

22á5 1á11 0á46 46á5 89

46á5 0á12 0á04 4á3 98

100 0á17 0á03 3á3 99

120 0á07 0á01 1á1 72

144 0á11 0á01 1á5 64

S2, Membrane-bound rPIN-a detected in the successive TX-114 phase

partitioning.

Table 2 In¯uence of pH on recombinant puroindoline-a (rPIN-a)

concentration (mg l)1), speci®c productivity (q; lg g)1 h)1) and

yield production (Y; lg g)1) during continuous culture no. 1 with the

44.8.26 clone

pH

rPIN-a

(mg l)1)

q rPIN-a

(lg g)1 h)1)

Y rPIN-a

(lg g)1)

% rPIN-a

membrane-

bound (S2)

5 1á16 0á96 19á3 87

6 0á13 0á11 2á20 55

7 0á04 0á03 0á62 98

A steady state was obtained at 29 °C when X = 60 g l)1 with a dilution

rate D of 0á05 h)1. S2, Membrane-bound rPIN-a.

Table 3 In¯uence of the temperature on recombinant puroindo-

line-a (rPIN-a) concentration (mg l)1), speci®c productivity

(q; lg g)1 h)1) and yield production (Y; lg g)1) during continuous

culture no. 1 with the 44.8.26 clone

Tempera-

ture (°C)

rPIN-a

(mg l)1)

q rPIN-a

(lg g)1 h)1)

Y rPIN-a

(lg g)1)

% rPIN-a mem-

brane-bound (S2)

29 0á06 0á05 0á98 67

29* 0á05 0á04 0á80 94

37 0á03 0á033 0á55 94

A steady state was obtained when X = 60 g l)1, at pH 6; with a

dilution rate D of 0á05 h)1. S2, Membrane-bound rPIN-a.

* 5 g l)1 casamino acids were added after 311 h when the temperature

was decreased to 29 °C.



production of rPIN-a seemed to be improved when the

culture was carried out with a low speci®c growth rate.

A third steady state allowed us to test the effect of the

addition of 0á01% TX-114 with a dilution rate of 0á06 h)1.

The concentration of rPIN-a increased to 1á58 mg l)1,

which was more than when the culture was carried out with

a dilution rate of 0á03 h)1 but without TX-114. Further-

more, when TX-114 was present in the culture medium,

most rPIN-a was in the soluble form in the culture

supernatant ¯uid. The speci®c productivity of rPIN-a was,

therefore, 1á58 lg g)1 h)1 and 1á6 times higher than the

productivity of rPIN-a when the ®rst continuous culture

was carried out with a dilution rate of 0á05 h)1 and at pH 5.

Production of recombinant puroindoline-aby continuous fermentation with favourableconditions

Continuous culture no. 3 was then carried out in order to

monitor the production of rPIN-a using the parameters

de®ned above. The temperature and pH were, respectively,

set at 29 °C and 5. The dilution rate was about D � 0á03 h)1

and 0á01% (v/v) TX-114 was added to the culture medium.

The 44.8.36 clone was used for this experiment because it

produced eight times more rPIN-a than the 44.8.26 clone by

fed-batch culture (data not shown). The dilution rate was ®rst

monitored from 0á003 to 0á012 h)1 during 10 h in order to

adapt the cells to methanol. This adaptation phase was run too

fast and the accumulation of formaldehyde required methanol

feeding to be stopped. TX-114 was supplied gradually to the

fermenter vessel inducing foam production, volume changes

and steady state interruptions that prevented the continuous

culture from being carried out with the desired dilution rate.

Nevertheless, three steady states were obtained with different

dilution rates: (i) D � 0á025 h)1; (ii) D � 0á033 h)1 and (iii)

D � 0á038 h)1. During the ®rst steady state, the concentra-

tion of rPIN-a increased from 3á3 mg l)1 at the end of the

adaptation phase to 13á8 mg l)1 (10 times higher than the

concentration of rPIN-a previously detected). The speci®c

productivity of rPIN-a reached 5á75 lg g)1 h)1. The next

two steady states ran with a higher dilution rate which could

explain the decrease in the production of rPIN-a to 7á1 and

1á52 mg l)1, respectively (Table 5).

However, more than 80% of the rPIN-a was secreted and

detected in the culture supernatant ¯uid when the dilution

rate was set to 0á025 h)1. The addition of 0á01% (v/v) TX-

114 inverted the ratio between the soluble and the mem-

brane-bound rPIN-a. A subcellular fractionation was per-

formed on 10 ml of the culture when the dilution rate was

0á038 h)1. Secreted rPIN-a represented only 31% of the

total rPIN-a and 22% of the rPIN-a was contained within

the perisplasm and cytoplasm of the yeast. Only 6% of the

rPIN-a remained insoluble and present within the pellet

fraction (Table 6).

DISCUSSION

For the ®rst time an rPIN-a was produced in fermentation

culture by the methylotrophic yeast P. pastoris. The aim of

the present study was to test different culture conditions

to improve the production of rPIN-a. The pYAM7SP8

expression vector was chosen because it allows cDNA fusion

to a secretory leader sequence which has often been used

successfully for the production and secretion of foreign

Table 4 In¯uence of the addition of TX-114 and the dilution rate

(D) on recombinant puroindoline-a (rPIN-a) concentration


lg g)1) during continuous culture no. 2 with the 44.8.26 clone

D (h)1) rPIN-a

(mg l)1)

q rPIN-a

(lg g)1 h)1)

Y rPIN-a

(lg g)1)

% rPIN-a mem-

brane-bound (S2)

0á03 1á38 0á69 23á0 86

0á05 0á43 0á35 7á16 91

0á06* 1á58 1á58 26á3 44

A steady state was obtained when X = 60 g l)1, T = 29 °C and at

pH 6. S2, Membrane-bound rPIN-a.

* 0á01% TX-114 was added after 270 h when the dilution rate was

0á06 h)1.

Table 5 Recombinant puroindoline-a (rPIN-a) concentration


lg g)1) during continuous culture no. 3 with the 44.8.36 clone

D (h)1) rPIN-a (mg l)1) q rPIN-a (lg g)1 h)1) Y rPIN-a (lg g)1)

0á025 13á8 5á75 230

0á033 7á10 3á89 118

0á038 1á52 0á96 25á3

Fermentation was carried out under favourable conditions. A steady

state was obtained when X = 60 g l)1, T = 29 °C, at pH 5, and with a

dilution rate D of 0á03 h)1. 0á01% TX-114 was added to the culture

medium.

Table 6 Protein measurement with the biuret reaction (mg) and

recombinant puroindoline-a (rPIN-a) measurement by enzyme-

linked immunosorbent assay (mg) in subcellular fractions after

yeast grinding

Protein content (mg)

Total rPIN-a

Secreted proteins 24 0á018

Membrane-bound proteins 46á1 0á023

Soluble proteins after cell breakage 228 0á013

Insoluble proteins after cell breakage 152 0á004

Subcellular fractionation was performed on 10 ml of continuous

culture no. 3 when the dilution rate was 0á038 h)1.



proteins (Laroche et al. 1994; De Klein et al. 1998; Inan

et al. 1999). The prohormome-processing KEX2 protease

allows the cleavage, on the C side, of a pair of dibasic

residues, Lys-Arg (Brenner and Fuller 1992). However, the

translocation folding, intracellular transport and secretion of

a 9-kDa rLTP have already shown the in¯uence of the

medium on the ef®ciency of KEX 2 (De Klein et al. 1998).

Because several studies have reported that Muts transfor-

mants sometimes result in an enhanced production of

foreign proteins compared with Mut� proteins (Cregg and

Madden 1987; Tshopp et al. 1987; Chirulova et al. 1997),

both AOX1 and HIS4 insertion were carried out. Among

the 28 clones giving a positive response by the immuno-

bloting detection, only two seemed to be Muts

. In fact, we

did not observe any signi®cant relationships between the

phenotype of transformants and the production of rPIN-a.

In several examples, improved recombinant protein expres-

sion yields were observed for multicopy clones (Romanos

et al. 1992; Cregg et al. 1993). These results are in good

agreement with our own results (data not shown). Two

Mut+ transformants were chosen to test the production of

rPIN-a and allowed the feeding rate to be more freely

controlled than a culture with a Muts strain.

During fed-batch fermentation, mature rPIN-a was

detected in the ®rst hours of methanol induction and then

disappeared suddenly. Several hypotheses can be proposed

to explain this observation. Proteases could be responsible

for the decrease in rPIN-a in culture supernatant ¯uid. The

use of a protease-de®cient strain has been shown to be a

successful approach to improve the production yield of

heterologous proteins in both Saccharomyces cerevisiae and

P. pastoris (Sander et al. 1994; Weiss et al. 1995). The

utilization of strain SMD1168 could lead to better results in

the production of rPIN-a. In the same way, the addition of

protease inhibitors to the culture media could increase the

production of rPIN-a. Nevertheless, rPIN-a did not remain

at the same concentration level despite the addition of

casamino acids during continuous fermentation in order to

prevent protease activity (Clare et al. 1991).

The changeover from the secreted to the membrane-

bound rPIN-a could be another hypothesis explaining this

decrease. In high density fed-batch fermentation most rPIN-

a remains membrane-bound during the culture. It had been

observed that PIN-a tends to form aggregates under acid

and high ionic strength conditions (Le Bihan et al. 1996).

Despite the presence of ®ve disulphide bridges, the

secondary structure of PIN-a is pH dependant and the

reduction in disulphide bonds leads to a signi®cant loss of

solubility. We have observed that the solubility of rPIN-a

increased at low pH; this is contradictory to previous

observations. The decrease observed after 43 h during fed-

batch culture was repeatable with all the clones tested in the

same culture conditions. Another hypothesis explaining the

decrease in rPIN-a in the culture supernatant ¯uid could be

based on non-detection by the antiserum against PIN-a

when rPIN-a forms aggregates with other cell proteins.

The speci®c growth rate had an important in¯uence on

the productivity yield of rPIN-a. Maximum productivity of

rPIN-a was observed during the initial period of slow

methanol feeding of the fed-batch culture. There is no

signi®cant relationship between the production yield of

foreign protein and the speci®c growth rate but, in a similar

way, a low speci®c growth rate seemed to improve the

production of a recombinant porcine follicle-stimulating

hormone (FSH) (Boze, personal communication).

The most striking result was obtained by the addition of

0á01% (v/v) TX-114 directly to the feeding medium. The

high af®nity of rPIN-a to polar lipids was a hindrance to

protein secretion. Phase separation under salt in¯uence for

non-ionic detergents is a simple and effective method for the

concentration of solubilized membrane proteins (Fricke

1993). The TX-114 phase partitioning allowed the remain-

ing cell-bound rPIN-a to be solubilized. Moreover, the

addition of TX-114 during fermentation was tested accord-

ing to the results, showing that the addition of 0á01% (v/v)

TX-100 to a feeding medium partially reduced the prote-

olysis of a urokinase-type plasminogen activator and

increased the secretion level (Tsujikawa et al. 1996). Finally,

TX-114 increased by 10-fold the production yield of rPIN-a

to 13 mg l)1 and inverted the ratio between secreted and

membrane-bound rPIN-a. This suggests that TX-114 may

perturb the adhesion of rPIN-a to the cell wall during yeast

growth and further secretion. Results of subcellular frac-

tionation have shown that an important part of rPIN-a was

still contained within the periplasm of P. pastoris. Whether a

secreted protein lodges in the periplasmic space, cell wall or

diffuses into the culture medium depends partly on the

size of that protein. The yeast cell wall strongly in¯uences

permeability in conventional yeasts and disulphide bridges

have been recognized to play a prominent role in determin-

ing the shape of the cell wall (Kidby and Davies 1970).

Thus, the cell wall represents an ion exchange ®lter with the

porosity being dependent on the cell wall charge (De Nobel

et al. 1990). Acidic conditions could increase the elasticity of

the cell wall and allow better secretion.

The aim of the production of rPIN-a was to provide large

quantities for the rPIN-a structural study. Contrary to all

expectations, the production yield was not the same as the

production yield obtained with other wheat seed cystine-rich

proteins (Klein et al. 1998). The production of rPIN-a could

be compared with the production yield (about 1 mg l)1) of

the heterologous membrane receptor secreted by P. pastoris(Cereghino and Cregg 2000). Our results allow a 10-fold

enhancement in the production yield of rPIN-a when

fermentation culture is carried out under precise conditions.

However, expected production yields were not reached. It



will be interesting to consider other expression systems in

order to increase the production of rPIN-a.

ACKNOWLEDGEMENTS

The authors would like to thank Dr L. Quillien (INRA

Nantes) for providing the polyclonal anti-PIN-a antibody,

P. Brignon (TEPRAL Strasbourg) for puri®ed PIN-a and

Dr Y. Laroche (Leuven Catholic University, Belgium) for

the pYAM7SP8 vector. This work was supported by the

MinisteÁre de l'Enseignement SupeÂrieur et de la Recherche

(grant MESR 97C0101) and N.I. was the recipient of a grant

from MENRT.

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Optimization of the wheat puroindoline-a production in Pichia pastoris

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