Cloning, Expression, and Characterization

of Recombinant Sweet-Protein Thaumatin II

Using the Methylotrophic Yeast
Pichia pastoris

Tetsuya Masuda, Shinobu Tamaki, Ryosuke Kaneko, Ritsuko Wada, Yuki Fujita,
Alka Mehta, Naofumi Kitabatake

Division of Food Science and Biotechnology, Graduate School of Agriculture,

Kyoto University, Uji, Kyoto 611-0011, Japan; telephone: + 81-(0)774-38-
3742; fax: + 81-(0)774-38-3743; e-mail: kitabatake@ kais.kyoto-u.ac.jp

Received 11 March 2003; accepted 19 June 2003

Published online 2 February 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10786

Abstract: Thaumatin, an intensely sweet-tasting protein, points (Van der Wel and Loeve, 1972). On a molar basis,
was secreted by the methylotrophic yeast Pichia pastoris. all of these forms are nearly 100,000 times sweeter than
The mature thaumatin II gene was directly cloned from Taq sucrose. Thaumatin I consists of a single-chain protein of
polymerase-amplified PCR products by using TA cloning
methods and fused to the pPIC9K expression vector that 207 amino acid residues and neither carbohydrates nor
contains Saccharomyces cerevisiae prepro a-mating factor unusual/modified amino acids are contained in the
secretion signal. Several additional amino acid residues molecule (Iyengar et al., 1979). The nucleotide sequence
were introduced at both the N- and C-terminal ends by of thaumatin II from cloned cDNA showed that thaumatin
genetic modification to investigate the role of the terminal II is translated as a prepro form with both a 22 amino acid
end region for elicitation of sweetness in the thaumatin
molecule. The secondary and tertiary structures of purified hydrophobic N-terminal extension and an acidic 6 amino
recombinant thaumatin were almost identical to those of acid-long carboxyl terminal extension (Edens et al., 1982).
the plant thaumatin molecule. Recombinant thaumatin II The deduced amino acid sequence of thaumatin II was
elicited a sweet taste as native plant thaumatin II; its different from that of thaumatin I at five sequence positions
threshold value of sweetness to humans was around (N46K, S63R, K67R, R76Q, and N113D) The three-
50 nM, which is the same as that of plant thaumatin II.
These results demonstrate that the functional expression dimensional (3-D) structure of thaumatin I was determined
of thaumatin II was attained by Pichia pastoris systems to consist of three domains (De Vos et al., 1985; Ogata et al.,
and that the N- and C-terminal regions of the thaumatin II 1992) (Fig. 1). The core domain consists of an 11-strand,
molecule do not play an important role in eliciting the flattered h-sandwich folded into two Greek key motifs.
sweet taste of thaumatin. B 2004 Wiley Periodicals, Inc. All h-strands are antiparallel, except the parallel N-termi-
Keywords: thaumatin; recombination; Pichia pastoris;
nal and C-terminal strands (1 – 53, 85 –127, and 178 –207,
sweet-tasting protein
domain I). The second domain is a large disulfide-rich region
(128 – 177, domain II). The third domain consists of a small
disulfide-rich region (54 – 68, domain III).
INTRODUCTION Since thaumatin is a protein and non toxic, and
constitutes the principal part of the fruit (at least 20% of
Thaumatin is a sweet-tasting protein isolated from the arils the aril dry weight), local people in West Africa have been
of Thaumatococcus daniellii Benth, a plant native to using thaumatin for its sweetness, flavor enhancement, and
tropical West Africa (Van der Wel and Loeve, 1972). synergistic properties in food products (Etheridge, 1994).
Analysis of the purified sweet proteins shows that Despite its advantages, the production of thaumatin from
thaumatin actually consists of six intensely sweet forms, T. daniellii, is limited and difficult. Alternative production
with two major components (thaumatin I and II) and four using genetic engineering has been attempted not only to
minor components (thaumatin III, a, b, and c). The ensure commercial use, but also to elucidate the unique
difference in the six variants can be attributed to isoelectric properties of the sweetness of thaumatin.
Although there have been numerous attempts to produce
thaumatin by using E. coli (Edens et al., 1982; Daniell et al.,
Correspondence to: Naofumi Kitabatake 2000), K. lactis (Edens and van der Wel, 1985), B. subtilis

B 2004 Wiley Periodicals, Inc.

 easily scaled-up for fermentation. These advantages are
extremely useful when a large quantity of products is
required, for example, for sensory analysis of the thaumatin
molecule in humans as some amount of recombinant
thaumatin is needed to assess taste.
Extensive studies have been devoted to confirm and
define the indispensable part of the thaumatin molecule for
elicitation of sweetness. Slootstra et al., 1995) prepared
monoclonal antibodies that cross-reacted with both thau-
matin and monellin, and suggested that epitopes of these
antibodies corresponding to amino acids 19 –29 and 77 –84,
in the thaumatin molecule, would be the important
determinants of the sweet taste. Chemical modification
studies have suggested that the basicity of thaumatin is not
the dominating factor in determining the intensity of its
sweetness (Shamil and Beynon, 1990; Van der Wel and
Bel, 1976). More recently, Kaneko and Kitabatake prepared
lysine-modified derivatives and carboxyl-group-modified
derivatives by chemical modification and concluded that
one side of the thaumatin molecule, i.e., the cleft-
containing side, is important for sweetness (Kaneko and
Kitabatake, 2001). Kim and Weickmann prepared over
40 different positions of thaumatin mutants to study the
Figure 1. Three-dimensional structure of thaumatin. The N- and C- properties of mutants in sweetness (Kim and Weickmann,
terminal residues are indicated by N and C, respectively. The figure was 1994). These mutated sites include the N-terminal amino
drawn using data for thaumatin (Ko et al., 1994) (PDB #1THV) and the
program MOLSCRIPT (Kraulis, 1991). acid (Ala), the loop polypeptide in domain II, and the
amino acids surrounding the Asp113, Lys97. The results
demonstrated that mutations that reduce the sweetness are
located on one side of the molecule, where domain II and III
meet (cleft-containing side). Despite extensive studies
(Illingworth et al., 1988), S. lividans (Illingworth et al., focused on domains II and III, both the N- and C-terminal
1989), S. cerevisiae (Lee et al., 1988), A. oryzae (Hahm and regions in domain I were not investigated in detail, except
Batt, 1990), and P. roquefortii (Faus et al., 1997), most of the attachment of acetylated N-terminal alanine residues,
them were not sufficient in protein yield or the form was which is produced intracellularly in yeast. Thus, the
inactive (non-sweet). Daniell et al. (2000) attempted to determinants of the sweet taste of thaumatin remain
express thaumatin II in E. co1i. This recombinant protein, controversial and obscure.
however, was produced in the form of insoluble inclusion In the present study, we attempt to construct and express
bodies. To obtain a soluble, correctly folded, and active a large quantity of thaumatin II by using the natural
thaumatin II, the renaturation procedure using a reduced/ thaumatin gene in the Pichia expression system for sensory
oxidized glutathione system is indispensable (Daniell et al., analysis of sweet protein. Through a protein-engineering
2000). Lee et al. (1988) introduced synthetic genes into approach, additional amino acid residues were introduced
S. cerevisiae to produce thaumatin by using yeast-preferred into both N- and C- terminal regions, and the roles of these
codons. Their group attempted to produce thaumatin by two regions in the elicitation of sweetness of thaumatin for
different methods and succeeded in obtaining a large amount humans were examined.
of sweet-tasting thaumatin (Weickmann et al., 1994).
Another expression system using A. niger var. awamori
has been tried with secretion of thaumatin in concentrations
of 5 –7 mgL 1 (Faus et al., 1998). MATERIALS AND METHODS
Recently, the methylotrophic yeast Pichia pastoris
has been developed as a host for the high-level production
Materials
of recombinant protein expression (Cereghino and Cregg,
2000; Goodrick et al., 2001; Hellwig et al., 2001). mRNA purification kit (QuickPrep micro mRNA purifica-
Pichia pastoris is a desirable expression system because tion kit) and cDNA synthesis kit (First-strand cDNA
it has a highly efficient and inducible AOXI promoter for synthesis kit) were both purchased from Pharmacia (Uppsa-
high-level expression of proteins, grows in minimal media la, Sweden). Ligation reaction was performed by using a
free of animal-derived contaminants, and achieves high cell ligation high kit (Toyobo, Osaka, Japan). Big Dye
densities in excess of 450 gL 1 wet cell weight, and is Terminator Cycle Sequencing Ready reaction for DNA

762 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 85, NO. 7, MARCH 30, 2004
sequencing was from PE Applied Biosystems (Warrington, acid, 0.5 g cobalt chloride, 20.0 g zinc chloride, 65.0 g
UK). Restriction enzymes were purchased from New ferrous sulfate-7H2O, 0.2 g biotin, and 5.0 mL, sulfuric acid.
England Biolabs, Inc. (Beverly, MA). Yeast extract, bacto
peptone, bacto tryptone, bacto agar, and yeast nitrogen base
Cloning of the Thaumatin Gene
(YNB) without amino acids were obtained from Difco
Laboratories (Detroit, MI). Thaumatin I and Thaumatin II A pyramidal-shaped fruit of Thaumatococcus daniellii kept
were purified from crude thaumatin powder as described at 80jC was powdered by mortar and pestle in the
previously (Kaneko and Kitabatake, 2001). The concen- presence of liquid nitrogen. The mRNA was purified by
trations of native thaumatin I and II were determined using mRNA purification kit. Briefly, about 0.5 mg of the
spectrophotometrically with a UV-160A spectrophotometer powder of the frozen tissue was dissolved in 0.4 mL, of
(Shimadzu Co., Kyoto, Japan) in 5 mM sodium phosphate extraction buffer. After the addition of 0.8 mL of elution
buffer, pH 7.0, using a molar extinction coefficient of q278 buffer, the mixture was centrifuged at 10,000 g for 1 min.
of 17,000 Mcm 1 (Van der Wel and Loeve, 1972). To obtain The supernatant was mixed with oligo (dT) cellulose sus-
q278, a mass of 22,209 Da (Iyengar et al., 1979) was used. pension and centrifuged at 10,000 g for 10 s. The preci-
Fruit of Thaumatococcus daniellii Benth, donated by San-Ei pitates were washed 5 times with 1 mL of high-salt buffer
Gen F.F.I. (Osaka, Japan), were kept at 80jC until use. and then washed twice with 1 mL of low-salt buffer. After
All other chemicals were of guaranteed reagent grade for washing, the supernatants were suspended in 0.3 mL of
biochemical use. low-salt buffer and applied to the MicroSpin column. After
centrifugation at 10,000 g for 5 s, the column was washed
with 0.5 mL of low-salt buffer three times and, finally, the
mRNA was eluted with 0.4 mL of elution buffer that was
Strains and Plasmids
preheated to 65jC by centrifugation (10,000 g, 5 s).
E. coli strains Top 10F (recA, endAl, HsdR), which were Ethanol precipitation was performed by mixing of 400 AL
used for cloning, transformation, and propagation of the ethanol, 10 AL glycogen solution, 40 AL potassium acetate
recombinant thaumatin plasmid, were obtained from solution, and 1 mL of 95% ethanol. First-strand cDNA
Invitrogen (Groningen, The Netherlands). Strain GS115 of synthesis was performed using a First-strand cDNA Syn-
Pichia pastoris was obtained from Invitrogen. Plasmid thesis Kit (Pharmacia, Uppsala, Sweden) as follows. Preci-
pCRR2.1-TOPOR which contains the linearized single pitated mRNA was dissolved in 20 AL DEPC water and
overhanging 3Vdeoxythymidine (T) residues and topo- heated at 65jC for 10 min. After being chilled on ice, iso-
isomerase I, was used for TA cloning. Plasmid pPIC9K, lated mRNA was reverse-transcribed using 1 AL of pd (N)6
which contains the inducible AOX1 promoter, a-factor primers, 1 AL of DTT solution, and 11 AL of bulk First-
prepo secretion signal, and a kanamycin selectable Strand cDNA Reaction Mix at 37jC for 1 h. The thauma-
resistance marker, was used as a yeast-E. coli shuttle tin gene was amplified by PCR using the first-strand
vector for thaumatin expression in the yeast. Both plasmids cDNA as a template, and 5V-GCCACCTTCGAGATCGT-
were from Invitrogen. CAAC-3V and 5V-CCTAGGGGCAGTAGGGCAGAA-3V as
primers (underlined Avr II site). The PCR reaction was con-
ducted using Taq polymerase for 1 cycle of 94jC for 1 min,
and 30 cycles of 96jC for 30 s, 67jC for 30 s, and 72jC for
Media Composition
1 min, and then 1 cycle of 72jC for 10 min. The PCR pro-
E. coli were cultured in LB medium (1% tryptone, 0.5% duct was analyzed by 1.2% agarose gel electrophoresis. The
yeast extract, and 1% NaCl). Pichia pastoris was grown in fresh Taq polymerase-amplified PCR product (1 AL) was
YPD (1% yeast extract, 2% peptone, and 2% dextrose) or immediately ligated into 1 AL of pCRR2.1-TOPOR vector.
BMGY. BMGY, a buffered glycerol complex medium, After being chilled on ice for 5 min, the PCR product was
consists of 100 mM potassium phosphate buffer, pH 6.0, transformed into Top 10F competent cells (Invitrogen)
1.34% YNB, 4  10 5% biotin, 2% peptone, and 1% mixed with 2 A1 of ligation mixture. The reaction mixture
glycerol. BMMY, a buffered methanol complex medium, was stored on ice for 30 min, was subjected to heat shock at
was identical to BMGY except that it contained 0.5% 42jC for 30 s, and then immediately transferred back to
methanol instead of glycerol. A minimal dextrose (MD) the ice. Then 250 AL, of SOC medium was added. Blue/
plate (1.34% YNB, 4  10 5% biotin, and 2% dextrose) was white screening was performed on LB plates containing
used for screening the Pichia transformants. Each liter of 50 AgmL 1 carbenicilin, 40 AL of 40 mgmL 1 X-gal, and
fermentation basal salts medium (FBS) consists of 26.7 mL 40 AL of 100 mM 1 IPTG. After incubation overnight at
phosphoric acid (85%), 0.93 g calcium sulfate, 18.2 g 37jC, some white colonies were observed. Plasmid DNA
potassium sulfate, 14.9 g magnesium sulfate-7H2O, 04.13g was isolated from E. coli by using the S.N.A.P. Miniprep Kit
potassium hydroxide, 40.0 g glycerol, and 4.35 mL PTMl (Invitrogen) and restriction analysis was performed by
trace salts. Each liter of PTMl trace salts contained 6.0 g digesting with EcoRI and Avr II at 37jC for 2 h. The DNA
cupric sulfate-5H2O, 0.08 g sodium iodide, 3.0 g manganese sequence was analyzed by an ABI 310 DNA sequencer
sulfate-H2O, 0.2 g sodium molybdate-2H2O, 0.02 g boric (Applied Biosystems) by using Big Dye Terminator Cycle

MASUDA ET AL.: CHARACTERIZATION OF RECOMBINANT SWEET-PROTEIN THAUMATIN II 763

Sequencing FS Ready Reaction Kit (Applied Biosystems) 0.5% every 24 h to maintain continuous induction. Protein
and Ml3 forward and reverse primers. The resulting plas- production was checked by sodium dodecyl sulfate-poly-
mid containing a thaumatin gene was named pCR2.1- acrylamide gel electrophoresis (SDS-PAGE) and Western
TOPO/thaumatin. blotting using antisera.

Construction of a Thaumatin Expression Vector Expression Thaumatin by Fermentor

pCR2.1-TOPO/thaumatin was digested with EcoRI and Large-scale fermentation was performed in a mini-jar
Avr II, yielding a fragment at about 0.6 kb, which encodes fermentor (M-100, Tokyo Rikakikai Co. Ltd., Tokyo, Japan)
the thaumatin gene. The digested fragments were isolated under the control of temperature, pH (FC-2000, Tokyo
from 1.2% agarose gels by using the Micropure and Rikakikai Co. Ltd., Tokyo, Japan), and dissolved oxygen
Microcon apparatus (Millipore Co., Bedford, MA). Purified (FM-2000, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The
thaumatin fragment (21.3 ng) and 160 ng of Pichia pastoris fermentation volume was 2 L. During fermentation, the
expression vector pPIC9K, which was predigested with temperature was maintained at 28jC with a heating unit
EcoRI and Avr II, were mixed with 1 Al, of Ligation high (Tokyo Rikakikai Co. Ltd., Tokyo, Japan) and a refrigerated
(TOYOBO) and incubated at 16jC for 1 h. Transformation circulating bath (RTE9, NESLAB, Newington, NH). The
was performed by using Top 10F competent cells concentration of dissolved oxygen was maintained at above
(Invitrogen) as described above. Positive colonies were 20% by use of an oxygen supply unit (MOS-25, Tokyo
screened by PCR and restriction enzyme treatment. DNA Rikakikai Co. Ltd., Tokyo, Japan). The pH was adjusted to
sequencing was performed as described above by using 5.0 through the addition of 25% ammonium hydroxide.
5VAOX1 primer and 3VAOX1 primers. The resulting plasmid Agitation rate is 900 rpm. Fermentation was initiated by
was named pPIC9K / thaumatin adding 50 mL, of the precultured yeast grown overnight in
BMGY medium to 1.0 L of fermentation basal salts medium,
containing 4% glycerol and 0.435% PTM1 trace salts. After
Electroporation of Pichia pastoris
complete comsumption of the glycerol in the medium, a
The pPIC9K / thaumatin plasmid was linearized by diges- glycerol fed-batch phase was initiated by the addition of
tion with Bgl II or Sac I overnight at 37jC. Pichia pastoris 50% glycerol containing 1.2% PTMl trace salts at a rate of
GS 115 was inoculated in a 5 mL of YPD medium overnight 18 mL L 1 h 1 with a peristaltic pump to increase the cell
and subsequently in 500 mL of YPD medium. Cells were biomass under restricted conditions. After 79 h, the wet cell
collected by centrifugation (1,500 g) and washed with sterile weight reached 200gL 1 at which time the glycerol-
water twice and finally washed with 1 M sorbitol. After methanol mixture was fed to initiate the induction of the
centrifugation, cells were resuspended in 1 mL of 1 M recombinant protein. During the first 4 h, glycerol feed rates
sorbitol. A mixture of 80 AL of GS115 concentrate and about and methanol feed rates were 10 mL L 1 h 1 and 4.0 mL
8 Ag of linearized vector was placed in 0.2 cm cuvettes where L 1 h 1, respectively. When the culture became adapted to
transformation was performed by electroporation with an methanol utilization (24 h after the methanol induction), the
Eppendorf electroporator 2510 (Hinz GmbH, Hamburg), set methanol feed rate was increased to 7.0 mL L 1 h 1 and the
at l,400 –1,700 V. Immediately after the transformation, glycerol feed rate was decreased to 7.5 mL L 1 h 1. Forty-
1 mL of ice-cold 1 M sorbitol solution was added to the eight hours after the methanol induction, the methanol feed
reaction cuvettes, and 250 AL of the mixture was spread on rate was adjusted to 11 mL L 1 h 1 and the glycerol feed was
separated MD (minimal dextrose) plates at 28jC for 2– 5 d. stopped. The methanol feed continued until the end of
fermentation. The total induction time was 132 h. The cells
were then recovered by centrifugation and the supernatant
Selection of the G418 Resistance Transformants
was analyzed by SDS-PAGE and Western blotting.
Multicopy transformants were selected on the basis of G418
resistance. Colonies on MD plates were picked up, resus-
PAGE
pended in 100 AL of sterile water, and screened on YPD
plates containing 2, 4, and 6 mgmL 1 of G418. SDS-PAGE was performed in a 13.5% gel according to the
methods of Laemmli (Laemmli, 1970). Native PAGE was
performed using a system of a 7.5% homogeneous native
Expression Thaumatin by Baffled Flask
polyacrylamide gel for the basic protein (Reisfeld et al.,
Each G418-resistant transformant was allowed to grow in a 1962). After electrophoresis, the gels were stained with
25 mL BMGY medium at 28jC until OD600 > 2. Cells were Coomassie Brilliant Blue R-250.
collected by centrifugation (1,500 g, 5 min) and resuspended
in 200 mL of BMMY medium. The cells were grown in a
Polyclonal Antibody Production
baffled flask with in an orbital incubator MIR-220R
(SANYO Electric Co., Ltd., Osaka, Japan) at 280 rpm for A polyclonal antibody against thaumatin I was prepared.
4 d at 28jC. Methanol was added to a final concentration of BALB/c mice were immunized with an emulsion mixture

764 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 85, NO. 7, MARCH 30, 2004
of thaumatin I with Freunds Complete Adjuvant. Three sample was incubated with dithiothreitol was (160 nM ) for
weeks later the mice were immunized with the mixtures of 15 min at 50jC and carboxamidomethylated with iodo-
purified thaumatin and Freunds Incomplete Adjuvant in the acetamide (320 nM ). Lys-C endoproteinase was added
same way. To check the production of the specific and the solution was incubated for 14 h at 37jC. The
antibodies, samples of sera were collected and tested by digestion samples were diluted 10 times with water and
ELISA. Sera was used for Western blotting without further freeze-dried by a centrifugal vaporizer (CVE-l00D, Tokyo
purification. The sera obtained reacted with thaumatin II as Rikakikai Co. Ltd., Tokyo, Japan). The dried sample was
well as with thaumatin I. dissolved in 500 AL of 50 mM sodium acetate buffer,
pH 5.0, containing 20 mM CaC12, and applied to an
anhydrotrypsin-agarose column (Takara Bio Inc., Kusatsu,
Western Blotting Shiga, Japan). The column was washed with 20 mM
sodium acetate buffer, pH 5.0, containing 20 mM CaCl2,
After SDS-PAGE, proteins were transferred to a PVDF and eluted with 5 mM HCl. The elutants were applied to a
membrane, Clear Blot Membrane-P, (ATTO, Tokyo, reverse-phase HPLC column (Cosmosil 5C18-AR-II: ODS,
Japan) by using a HorizBlot apparatus (Model: AE-6677, 4.6150 mm; Nacalai Tesque, Kyoto, Japan). The flow rate
ATTO, Tokyo, Japan) at 2.5 mAcm 2 for 1.5 h. Detection was 0.42 mLmin 1 and the elution was performed in the
was performed by using mouse anti-thaumatin sera as the presence of 0.1% trifluoroacetic acid (TFA) with a linear
primary mouse antibody and biotinylated anti-mouse anti- gradient of 10 – 90% acetonitrile. The purified fragments
body (Vectastain ABC-AP KIT; Vector, Burlingame, CA) were mixed with a-cyano-4-hydroxycinnamic acid, and the
as the second antibody. The membrane was incubated with mixture was centrifuged at 10,000 rpm for 5 min. The
primary antibody solution at room temperature for 30 min supernatant was analyzed by MALDI-TOF-MS (Voyager
and soaked in washing buffer (5% skim milk, 20 mM RP Biospectrometry Workstation, PerSeptive Biosystems,
sodium phosphate buffer, pH 7.5 containing 150 mM NaCl, Inc., Framingham, MA).
and 0.2% Tween 20) for 5 min 3 times. After incubation
with the second antibody, the membrane was washed with
T-TBS (25 mM Tris-HCI, pH 7.4 containing 0.14 mM NaCl Circular Dichroism Spectra
and 0.05% Tween 20) for 5 min 3 times. Enhancement was
achieved by use of the ABC-AP reagent supplement in the Circular dichroism (CD) spectra were recorded with a J-720
ABC-AP kit and NBT/BCIP stock solution (Roche Diag- spectropolarimeter (Jasco Co., Tokyo, Japan) at 25jC in
nostics Gmbh, Manheim, Germany). 5 mM sodium phosphate buffer, pH 7.0. Far-UV CD spectra
were obtained at a protein concentration of 4.5 AM with a
l-mm cell at wavelengths from 250– 200 nm. Near-UV
spectra were recorded at a protein concentration of 4.5 AM
Purification of Recombinant Thaumatin
with a l-cm cell at wavelengths from 250 –350 nm. The data
The supernatant of the culture was dialyzed against 5 mM was collected 3 times and are given as the average mean
sodium phosphate buffer, pH 7.0. Subsequently, the residue ellipticity.
dialysate was applied to the Cellulofine CM-ION Ex-
changer C-500 (Seikagaku Kogyo, Tokyo, Japan). The
recombinant thaumatin was eluted stepwise with 5 mL of Fluorescence Spectra
50 mM, 200 mM, and 1M NaCl.
A trytophan fluorescence spectrum was recorded at 25jC
with a fluorescence spectrophotometer (F-3000; Hitachi,
Ltd., Tokyo, Japan). The protein concentration was
N-terminal Sequence Analysis
1.125 AM. The excitation wavelength was 295 nm and
N-terminal sequence analysis was performed in a gas-phase the emission wavelength ranged from 300– 400 nm.
sequencer (Procise 492, PE Biosystems, Foster City, CA)
using the Edman degradation methods. Two hundred pico
molar of samples, which was transferred to PVDF mem- Sensory Analysis
brane, was analyzed.
The sweetness threshold of the samples was evaluated by
means of a triangle test for taste absolute threshold (Kanko
and Kitabatake, 2001). Five milliliters of test solution,
C-terminal Sequence Analysis
which were prepared in 5 mM sodium phosphate buffer,
The C-terminal sequence was determined by using matrix- pH 7.0, were added to a paper cup. As a control, 5 mM
assisted laser-desorption ionization time-of-flight mass sodium phosphate buffer, pH 7.0, was used. Six subjects
spectrometric (MALDI-TOF-MS) analysis. The lyophi- participated in this trial and attempted to test the sample
lized recombinant thaumatin (200 Ag) dissolved in 100 AL solutions in order of concentration from lower (10 nM) to
of 8M urea and 0.1M ammonium bicarbonate buffer. The higher (500 nM ).

MASUDA ET AL.: CHARACTERIZATION OF RECOMBINANT SWEET-PROTEIN THAUMATIN II 765

RESULTS

TA Cloning of Thaumatin Gene and Construction

of Expression Vector
Thaumatin gene was amplified by PCR and amplified PCR
products were directly cloned into the TOPO-PCR2.1
vector that had an overhanging thymidine residue. DNA
sequencing was performed and the sequence was identical
to that of thaumatin II as determined by Edens et al. (1982).
This plasmid was digested with EcoRI and Avr II and the
fragment was ligated into the EcoRI and Avy II sites of the
pPIC9K yeast-shuttle vector. PCR analysis using thauma-
tin-specific primers (M13 reverse and forward primers) and
Figure 3. Time course of fermentation profile of thaumatin-expressing
restriction analysis (digested with EcoR1 and Avr II) were
P. pastoris. OD280 (closed circle), wet cell weight (open circle), glycerol
performed and approximately 0.6 kb was observed in both and methanol feeding (open and closed triangles), and oxygen (open
analyses (Fig. 2). square) are plotted according to incubation time.

Expression of Recombinant Thaumatin

Protein expression was initiated by the addition of addition of 99:71 methanol containing PTM trace salts
methanol. We used a fermentor that was equipped with a (12 mL L 1). After 132 h of induction—211 h of
controller to maintain a given value of dissolved oxygen, fermentation in total—the culture medium was centrifuged
pH, and temperature. The secretion level of protein (1500 g, 20 min) to collect cells. Analysis of the
observed by absorbance at 280 nm and the wet cell weight supernatant by SDS-PAGE and Western blotting found
is shown in Figure 3. After 79 h of fermentation, the wet that recombinant thaumatin was secreted into the culture
cell weight reached 200 gL 1. At that point, methanol medium. Purification of recombinant thaumatin was per-
feeding was started. Protein expression was induced by formed by CM-ion exchange column chromatography. The
single major peak eluted with a 0 –0.5 M NaCl gradient was
collected. Both SDS-PAGE and Western blotting of the peak
fraction showed a single band with the same mobility, which
had a slightly higher molecular size than that of authentic
thaumatin (Fig. 4). The expression level was approximately
25mg per liter of culture.

Figure 4. SDS-PAGE and Western blotting of thaumatin. (a) SDS-PAGE

was performed on 13.5% gel and stained with Coomassie Brilliant Blue.
Lane 1, MW marker; Lane 2, thaumatin I; Lane 3, thaumatin II; Lane 4,
culture supernatant; Lane 5, purified recombinant thaumatin II. (b) Western
Figure 2. Ligation of the thaumatin gene with yeast-shuttle vector blotting analysis of thaumatin was performed by using mouse anti-
pPIC9K. The thaumatin gene predigested with EcoR I and Avr II was thaumatin I sera as a primary mouse antibody, biotinylated anti-mouse
ligated into the EcoRI and Avr II sites of pPIC9K yeast-shuttle vector. antibody as a second antibody. Detailed conditions are given in the text.

766 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 85, NO. 7, MARCH 30, 2004
Characterization of Recombinant Thaumatin
Determination of the N-terminal sequences of recombinant
thaumatin was performed. In addition to that of authentic
thaumatin, Ala and Leu residues were observed at the
N-terminal of the recombinant thaumatin. Three additional
C-terminal residues (Pro-Arg-Ala) were also observed in the
purified Lys-C endoproteinase fragment by MALDI-TOF-
MS analysis. The results of the CD spectra in the far-UV
region indicated that the absolute ellipticity at 220 nm was
almost same among the thaumatin I, thaumatin II, and
recombinant thaumatin II (Fig. 5A). These results demon-
strated that the secondary structure was maintained despite
the introduction of additional amino acids at the N-
and C- terminal regions. No detectable differences in the
dichronic contents in the near-UV spectra were observed Figure 6. Fluorescence spectra of thaumatin derivatives. The trytophan
between the recombinant and authentic thaumatin mole- residues in thaumatin I (dotted line), thaumatin II (broken line), and
cules (Fig. 5B). These results suggest that no disruption of the recombinant thaumatin II (solid line) were excited at 295 nm and the
configuration around the aromatic amino acid region emission spectra were recorded at 25jC.
was observed and that the tertiary structures of thauma-
tin I, thaumatin II, and recombinant thaumatin II were The changes in the environment around the Trp residues
almost identical. were examined by fluorescence spectra (Fig. 6). Fluores-
cence spectra showed that recombinant thaumatin had the
same emission maximums as those of native thaumatin I
and II while the fluorescence intensity of recombinant
thaumatin II was slightly different from those of thaumatin
I and II. This indicates that recombinant thaumatin II does
not differ remarkably in its secondary and tertiary
structures from those of thaumatin I and II. The attachment
of additional N- and C-terminal residues had little influence
on the structure of the thaumatin molecule.

Sensory Analysis in Humans

The threshold of the detection of sweet taste of recombinant
thaumatin in humans was around 50 nM. In our sensory
analysis, four of six subjects detected the sweetness of
thaumatin I at 50 nM or less, and five of six subjects
detected the sweetness of thaumatin II at 50 nM. Five of six
subjects detected the sweetness of recombinant thaumatin
II at 50 nM just as well as that of native thaumatin II
(Table I). These results indicate that the N- and C-terminal

Table I. Threshold of sweetness of thaumatin derivative in humans.a

nM Thaumatin I Thaumatin II Recombinant Thaumatin II

10 0 0 0
20 2/6 0 0
50 2/6 5/6 5/6
100 2/6 1/6 0
200 0 0 1/6
500 0 0 0
Mean F SD 57 F 32 58 F 19 75 F 56
Figure 5. Far-UV and near-UV CD spectra of thaumatin derivatives. The
a
CD spectra of thaumatin I (dotted line), thaumatin II (broken line), and The sweetness threshold of samples was evaluated by means of a
recombinant thaumatin II (solid line) were measured in 5 mM sodium triangle test for taste absolute threshold. Six subjects participated in the
phosphate buffer, pH 7.0, as described in the Materials and Methods test and examined thaumatin I, thaumatin II, and recombinant thaumatin II
section. (a) Far-UV CD spectra and (b) near-UV CD spectra. The data was prepared in 5 mL of 5 mM phosphate buffer, pH 7.0. As a control, 5 mM
collected three times and is given as the average mean residue ellipticity. sodium phosphate buffer, pH 7.0, was used.

MASUDA ET AL.: CHARACTERIZATION OF RECOMBINANT SWEET-PROTEIN THAUMATIN II 767

residues do not affect the sweetness of thaumatin and that expressed synthetic thaumatin genes in S. cerevisiae and
the N- and C-terminal regions of thaumatin are not directly found that yeast-produced thaumatin has a blocked amino-
related to elicitation of a sweet taste in thaumatin. terminal alanine. They investigated the difference between
the plant and yeast-produced thaumatin and found no
significant difference in the biological activity and sweet-
DISCUSSION
ness threshold. Our results proved that besides attachment at
The production of thaumatin has been attempted by using the N-terminal region, attachment at the C-terminal
many different microorganisms and most of them em- region also has little influence on the sweetness of thau-
ployed a synthesized DNA-encoding sequence for the matin. Furthermore, the ends of the h-strands A (N-ter-
thaumatin molecule with optimized codon usage. In the minal) and K (C-terminal) regions are not related to the
present study, we cloned a natural thaumatin gene and no elicitation of sweetness by thaumatin.
insertion and deletion were observed in comparing it with The structural requirements for elucidating the sweetness
the reported sequence (Edens et al., 1982). Edens et al. of thaumatin molecules were investigated from the
(1982) attempted molecular cloning of the natural thauma- perspective of chemical modification (Shamil and Beynon,
tin gene and found that thaumatin II contained an extra 1990; Van der Wel and Bel, 1976). The conclusions were
acidic 6 amino acid carboxyl terminal extension besides 22 that the positive charge of the thaumatin molecules is not
hydrophobic residues as the amino acid amino-terminal necessary for eliciting the sensation of sweetness. However,
secretion signal. They investigated the processing of these previous studies did not identify the position of the
thaumatin by expressing different types of thaumatin amino acid residues modified by reagent, and no specific
(preprothaumatin, prethaumatin, and prothaumatin) in residues for sweetness were elucidated. A site-directed
E. coli and S. cerevisiae, and concluded that both the mutagenesis study has also been performed. It demonstrated
signal sequence and the C-terminal extension played a that mutation at Lys67, Lys137, or Tyr169 reduced
significant role in thaumatin synthesis (Edens et al., 1984). sweetness fivefold. Thus, the determinants of the sweet
For this reason, we designed our study to clone the mature taste of thaumatin remain controversial and obscure. More
thaumatin II gene under S. cerevisiae a-factor prepro signal recently, Kaneko and Kitabatake (2001) investigated 5 of 11
peptide in a yeast shuttle vector that contains a highly lysine residues for elucidation of sweetness by methods of
efficient and inducible AOX1 promoter. The effect of chemical modification. They demonstrated that one side of
introducing additional amino acid residues at both the N- the thaumatin molecule, the cleft-containing side, is
and C-terminal end on the sweetness of the thaumatin important for the elicitation of the sweetness of thaumatin,
molecule was also investigated. Despite designing the suggesting the importance of the positive charge of the local
introduction of six amino acid residues (Tyr-Val-Glu-Ser- area on the molecular surface. These results are consistent
Ala-Leu) at the N-terminal, the resulting product showed with our present results, because both the N- and C-terminal
that only two residues (Ala-Leu) were attached. This regions are separate from the cleft-containing side.
confirmed that the processing of the N-terminal regions Although seven proteins, thaumatin (Van der Wel and
in Pichia pastoris occurred between Ser and Ala. During Loeve, 1972), monellin (Morris and Cagan, 1972), pentadin
fermentation, some cells might lyse releasing significant (Van der Wel et al., 1989), mabinlin (Liu et al., 1993),
amounts of proteinases from all parts of the cell. Moralejo curculin (Yamashita et al., 1990), brazzein (Ming and
et al. (2000) attempted to isolate an aspergillopepsin A- Hellekant., 1994), and lysozyme (Masuda et al., 2001) were
defective mutant of Aspergillus awamori containing an known as eliciting a sweet taste, no common features have
insertion in the pepA gene and succeeded to increase in been observed among these proteins either in terms of
thaumatin yields. Using Pichia systems, approximately amino acid sequences or in tertiary structures.
25 mg of purified thaumatin could be obtained per liter of Thaumatin has a high degree of homology to pathogen-
culture. By using protease deficient strains such as SMD related proteins and maize inhibitor, while the biological
l,168 or remove of N- and C-terminal extra amino acids, the function of thaumatin in the plant remains unclear.
yields of active protein would be improved. Weickmann Together with the chemical modification studies, muta-
et al. (1994) was able to secrete high levels of sweet-tasting genesis studies using a Pichia expression system provide
thaumatin (300 mgL 1) by using the synthetic thaumatin not only sufficient amounts of samples for sensory analysis,
gene for the yeast-preferred codon. Nevertheless, we used but also important implications for understanding the
natural thaumatin gene directly; we obtained enough sweet- perception of the sweet taste of thaumatin.
tasting thaumatin molecules for sensory analysis. This result
indicates that the powerful AOX1 promoter in Pichia
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