J. Biol. Macromol., 9 (2), 63-72 (2009)

Regular paper
Attempts to express recombinant Baker’s yeast glutamate decarboxylase in Saccharomyces
cerevisiae and Escherichia coli
Mariko Kanbara*, Akiko Fukuyama*, Yoshie Kuronuma*, Atsuko Shinmori, Mariko Ide,
Kumiko Machida, Hitomi Kiyohara, Miyuki Hayashi, Yoko Nitta, and Hiroshi Ueno§
Laboratory of Applied Microbiology and Biochemistry, Nara Women’s University, Nara 630-8506,
Japan
Received November 21, 2008; Accepted May 12, 2009

GABA-synthesizing enzyme, GAD**, in Saccharomyces cerevisiae was expressed in S.
cerevisiae and E. coli. S. cerevisiae expression system gave specific activity of 60
nmol/min/mg, which was 9-fold higher than the value obtained from the parental strain. S.
cerevisiae system expressing C-terminal His-tag GAD (GAD-His6) gave the specific activity of
27 nmol/min/mg. SDS-PAGE results of the crude extract followed by Western blot analysis
with the use of antibody against His-tag suggested that proteolysis took place. Because ofthis susceptibility to proteolysis, an expression of GAD-His6 in E. coli was attempted, where
GAD-His6 was found in both soluble and insoluble fractions. The soluble GAD-His6 in the
crude extract exhibited an enzyme activity of 10 nmol/min/mg with a major band at 69 kDa.
The expression systems can be a useful tool to study structure-function relationship of GAD,
i.e., preparation of antibody.
Key words: GABA, GAD, yeast, expression

γ-Aminobutyrate (GABA) is anamino acid found in animals, plants, andmicroorganisms. In higher animals, GABA
functions
as
a
major
inhibitory
neurotransmitter.
In addition, GABA isreported to participate various physiologicalactivities such as reduction of high bloodpressure, diuretic effect and induction of
growth factors[1-3].
Because of
beneficial physiological roles, various*Those authors contributed equally§
To whom correspondence should be
addressed: E-mail: hueno@cc.nara-wu.ac.jp,
Phone: +81-742-20-3493, Fax:
+81-742-20-3448

commercial foods that contain GABA as aningredient have been developed, where thefermentation is the main tool for the GABA
production. Among the microorganisms,
yeast is the most popular microorganism used
for production of foods containing GABA,
thus it is an ideal microorganism toinvestigate GABA-production mechanism.

Abbreviations: GABA, γ-amino butyricacid; GAD, glutamate decarboxylase;
TTBS, Tris-buffered saline with Tween-20**
Protein product of GAD1 should be called
Gad1p; however, because of the common
name for glutamate decarboxylase, theexpressed protein is called GAD in this text.

Expression of yeast glutamate decarboxylase

64

GABA is synthesized by glutamate
decarboxylase (GAD, E.C. 4.1.1.15) thatcatalyzes decarboxylation reaction of
L-glutamate [4]. GAD is found in various
species with different properties [5]. GAD
found in Escherichia coli has two isoforms,
GADA and GADB, both of which have thehexameric form and are active in the acidic

Scheme I.

conditions. When decarboxylation reaction
is in progress, GAD utilizes one molecule ofproton (H+) together with L-Glu to give one
molecule of GABA. This suggests that
GAD acts to neutralize the acidified
intracellular pH of the cell that has been
exposed to the acidic environment. Hence,the property of acid resistance of E. coli may

Schematic representation of plasmid designs

Kanbara, M. et al.

owe to the decarboxylation reaction [6, 7].
Several forms of GAD, GAD1 to 5, are foundinplantspiecesand theyhave
2+
Ca /calmodulin binding domain at the
C-terminal. The role of calmodulin binding
domain has been extensively studied andthought to be related to the plant growth [8].
The plant GADs are reported to be dimeric or
hexameric enzymes [9, 10]. Mammals have
two distinct genes for GAD that produce two
isoforms, GAD67 and GAD65. In brain
and nerve cells, while both isoforms are
expressed within the same cells, very little
information on the specific role(s) for each of
the isoforms is available. Previous studies
using knock-out mouse have given someclues for the individual roles of isoforms, inwhich GAD67 knock-out mice being bornwith cleft palate died within a day after thebirth whereas GAD65 knock-out micesurvived with an increased tendency having
seizures [11, 12]. Despite of these clearly
different functions for mammalian GAD
isoforms, their enzymatic function in
producing GABA from L-glutamate appears
to be the same.
Saccharomyces cerevisiae, a common
yeast employed for various food productions,expresses GAD protein that is encoded by
GAD1 gene. The role of GAD in yeast is
not clearly understood; however, a reportsuggests that GAD is involved in theresistance for oxidative stress [13]. As
shown in the recently available genome
database of S. cerevisiae, an open reading
flame, YMR250w, has been assigned for
GAD1 which is on chromosome 13. With
this information, we have cloned GAD1
cDNA and incorporated into expression
vectors. After the transformation of either S.
cerevisiae or E. coli, the expression profilesof the host cells were characterized and thebiochemical study on the obtained GAD was
carried out.

65

MATERIALS AND METHODS
Plasmids and host strains - Plasmid
pYES2 was obtained from Invitrogen(Carlsbad, CA). The pYES2 incorporated
with GAD1 (pYES2-GAD) was used totransform GAD knockout yeast (clone ID
20835) obtained from Open Biosystem(Huntsville, AL). The GAD knockout yeast
was created from strain BY4743 (MATa/αhis3∆1/his3∆1, leu2∆0/leu2∆0, MET15/
met15∆0, lys2∆0/LYS2, ura3∆0/ura3∆0) by
the Saccharomyces genome deletion project(Open Biosystem).
Plasmid pET21apurchased from Novagen (Madison, WI) wasused for the expression of the His-tag GAD
in E. coli strain BL21(DE3).
Typicalplasmid construction was summarized in
Scheme I.
Cloning and expression of GAD in
S. cerevisiae - The sequence of GAD1 was
obtained from Saccharomyces genome
database(http://www.yeastgenome.org/).
GAD cDNA was amplified by PCR withprimer pairs, 5’ GAA GTA TCT CGA GTAACA GTG ATG CTC 3’ and 5’ TTA TAG
GCG CTA GCC CTT TCA CC 3’. The
PCR product was cloned into pT7BlueT
vector purchased from Novagen and inserted
into the XhoI-PstI site of pBluescript to form
pBS-GAD.
Then, the pBS-GAD wasdigested with KpnI-SacI and inserted into thesame site of pYES2 to form pYES2-GAD.
In order to accommodate C-terminal His-tag,
the cDNA of GAD in pBS-GAD was mutatedto replace the stop codon with XbaI site byusing Quick Change mutagenesis kit(Stratagene, Cedar Creek, Texas).
The
primer pairs for the mutation were 5’ GGATGA CCT TTT CAT CTA GAC ATG TTC
CTC 3’ (XbaI restriction site) and 5’ GAGGAA CAT GTC TAG ATG AAA AGG TCA
TCC 3’ (XbaI restriction site). Then, thepPICZaB plasmid (Invitrogen) was digested
with KpnI and XbaI and the KpnI-XbaI

66

Expression of yeast glutamate decarboxylase

segment was inserted into this plasmid tocreate pPICZ-GAD-His6.
The pPICZ-GAD-His6 was digested
and inserted into KpnI-SacI site of pYES2 toobtain pYES2-GAD-His6.
Restriction
digestion and partial sequencing of theplasmid was carried out in order to confirm
the successful incorporation of the insert
DNA. Then, the plasmids were introduced
into the GAD1 knock out yeast by means ofelectroporation (BioRad, Richmond, CA).
The transformants on the plates containing
minimal synthetic medium without uracil(SC-U) were selected for the uracil
prototrophy.
S. cerevisiae carrying
pYES2-GAD or pYES2-GAD-His6 weregrown in a medium, containing yeast extract,
peptone and glucose (YPD), at 28 °C in ashaking incubator rotating at 200 rpm forovernight. The cells were centrifuged andresuspended
intogalactose-containing
medium (YPG) for the target protein
induction. After the culture flasks being
shaken for several hours at 28 °C, the cellpellets were collected by centrifugation andresuspended in the lysis buffer containing 1
mM 2-aminoethylisothiouronium bromidehydrobromide,
0.5
mM
pyridoxal
5’-phosphate (PLP), 0.1 mM dithiothreitol,
60 mM potassium phosphate buffer (pH 7.1)
and the protease inhibitor cocktail (Roche).
The cells suspended in the lysis buffer was
disrupted with a beads-beater (Biospec
Products, Kyoto, Japan) for 1 min and cooledfor 2 min, which was repeated 7 times.
During the cell lysis, the temperature wasmaintained not to exceed 10 °C bysurrounding the container with ice water.
The lysate was centrifuged at 10,000 x g for
20 min at 4 °C and the supernatant was used
as a crude enzyme extract.
Cloning,
expression
andpurification of GAD in E. coli - Prior to theinsertion of GAD1 cDNA into pET21a vector,
KpnI site was introduced into the vector byusing Quick Change mutagenesis kit(Stratagene). As a result, pET21a vector
should have KpnI and SalI sites toaccommodate the insert DNA. The primer
pair sequences for KpnI site were 5’ GGT
GGA CAG CAA AGG TAC CGC GGA TCC
G 3’ (KpnI restriction site) and 5’ CGG ATC
CGC GGT ACC TTT GCT GTC CAC C 3’
(KpnI restriction site).
Meanwhile apBS-GAD plasmid, in which the stop codon
for GAD ORF was removed, was digested
with KpnI and SalI to obtain the insert DNA.
Both pBS-GAD and pET21a were ligated
together to construct pET21a-GAD-His6,
which has His-tag at its C-terminus region.
The recombinant plasmid was confirmed by
the restriction digestion mappings and partial
sequencing. The plasmid was introduced
into E. coli BL21(DE3) strain by the calcium
chloride method.
E. coli transformant having
pET21a-GAD-His6 was grown at 37 °C for
overnight in LB medium containing 50 µg/ml
ampicillin.
This overnight culture wasdiluted in the fresh LB medium with 50µg/ml ampicillin to make initial OD at 0.05
and was grown at 37 °C until OD at 600 nm
reached 0.6. The GAD protein expression
was induced by adding 0.1 or 1mM
isopropyl-β-D-thiogalactoside (IPTG) andthen continued shaking the culture flasks at
18, 28, or 37 °C. After the centrifugation,
the cell pellet was collected and suspended
with an equal amount of PBS, constituted
with 0.2 g/l KCl, 0.2 g/l KH2PO4, 8 g/l NaCland 1.15 g/l Na2HPO4. The suspension was
treated with ultrasonication to disrupt thecells, and centrifuged at 10,000 x g for 20
min at 4 °C to obtain a crude enzyme extract.
The insoluble fraction was solubilized in a
denaturing buffer containing 1% SDS, 50
mM sodium phosphate, pH 8.0, and 0.3 M
NaCl, and was applied to a resin of nickel
based- (GE Healthcare, Buckinghamshire,UK) and cobalt based- (Clontech, MountainKanbara, M. et al.

View, CA) immobilized metal affinity
chromatography for protein purificationaccording to the manufacturer’s instructions.
Enzyme activity assay - The crude
enzyme extract was applied to a desalting
column to remove small molecular weight
impurities that affect fluorescent detection ofo-phthalaldehyde (OPA)-derivatized GABA.
At the same time, the buffer in the crude
enzyme extract was replaced with 50 mM
potassium phosphate, pH 7.0. GAD activity
was estimated by measuring the amount of
GABA produced during the assay under thecondition as described previously [14]. The
assay mixture, contained 400 µl enzyme
solution, 20 µl of 1 M L-glutamic acidneutralized to pH 7 with NaOH, 20 µl of 10
mM PLP neutralized to pH 7 with NaOH,
160 µl of 1 M sodium acetate buffer, pH 4.6,
and 400 µl H2O, was incubated at 37 °C for
30 minutes and the reaction was terminatedby adding 50 µl of 60% perchloric acid
(PCA). As a control, the same mixture wasprepared but PCA was added at time 0.
After the termination, aliquots of the mixture
were applied on an HPLC (Shimadzu, Kyoto,
Japan) equipped with the post-column
derivatization system that delivered OPA. A
CX Pak column (Shodex, Tokyo, Japan)

67

packed with strong cation exchange resin wasused to separate GABA and the abundantamount of L-glutamate by eluting with 0.2 N
sodium citrate buffer, pH 5.12, as describedby Spackman, et al. [15, 16] at a flow rate of
0.3 ml/min. The eluted solution of the CX
Pak column was mixed with OPA solution
and the derivatized amino acids weremonitored by a fluorescence detector(Shimadzu) set for excitation at 355 nm andemission at 440 nm. The amount of GABA
produced was calculated by comparing thepeak area of the examining solution with thatof the standard GABA solution of known
concentration. The protein concentration in
the enzyme solution was estimated by using
BioRad protein assay.
SDS-PAGE and Western blotting During the purification process, protein
purity was evaluated by carrying out
SDS-PAGE and Western blot analyses [17].
SDS-PAGE gel was electrically blotted to a
PVDF membrane on a BioRad semi-dry
blotter for 1 hr with the use of CAPS buffer,
pH 11. The membrane was treated withskim milk for 1 hr, washed with TTBS buffer(20 mM Tris, 154 mM NaCl, 0.05%
Tween-20, pH 7.4), and incubated withhorseradish peroxidase-conjugated mouse

68

Expression of yeast glutamate decarboxylase

monoclonal antibody against a His-tag andthe immuno-positive bands were visualizedwith ECL Western blotting detection reagent(GE
Healthcare)accordingtothemanufacturer’s instructions.
After thetreatment with ECL reagents, the membrane
was covered with plastic bag, heat sealed,
and placed on a Polaroid film (Type 667) in a
cassette (Invitrogen) to expose protein bands.

RESULTS AND DISCUSSION
Expression of recombinant GAD
and His-tag GAD in Saccharomyces
cerevisiae - A S. cerevisiae strain wastransformed with an expression plasmid,
pYES2-GAD, and cultured in YPD medium.
The recombinant GAD protein production
was initiated by replacing YPD medium with
YPG that contained galactose as a carbon
source. After cells were harvested, lysed,
and centrifuged, the soluble fraction wascollected and its aliquots were measured for
GAD activity. Specific activity of about 60
nmol/min/mg was obtained from the sample
of 4 h induction in YPG at 28°C. This

Fig. 1. Western blot of insoluble
fraction of S. cerevisiae transformed
with pYES2-GAD-His6.
Antibody used was horseradish
peroxidase-conjugatedmousemonoclonal antibody raised against
His-tag.

value is about 9-fold greater than thatobtained from the parental strain, BY4743,
which lacks plasmid (Table 1). When bothcell lysates from the recombinant andoriginal strains were subjected for
SDS-PAGE analysis stained with Coomassie
Brilliant Blue R-250 (CBB), they exhibitednearly identical band patterns at around 66
kDa as expected for GAD protein, however,the staining of the 66 kDa band for therecombinant strain was weak and theexpression level of GAD protein was notextensive enough for the biochemical studies.
It has been desired to have antibody
against yeast GAD in order to carry out
detailed biochemical studies, however,antibody is not available. Hence, at the
moment, it is not possible to carry out theWestern blot study. When the sequences
between yeast and mammalian GADs werecompared, there were some homologies thatsuggested some possibilities of cross-reactionof available GAD antibodies. Hence, wehave tried using antibodies against
mammalian GADs; but we were unable todetect any band with these antibodies.
Because weak expression of GAD in theyeast expression system and lack of theimmuno-detection method have discouraged
us to further characterizing the above
described system.
Thus, we have constructed a
pYES2-GAD-His6 by inserting His-tag
segment of pPICZaB into pYES2-GAD.
pYES2-GAD-His6 plasmid was introduced
into the competent cell of S. cerevisiae. The
colonies carrying pYES2-GAD-His6 plasmid
were isolated and subjected for His-tag GAD
protein production by the induction withgalactose.
The optimal expression of
His-tag GAD protein was achieved by 6 h
induction in YPG at 28 °C with specificactivity of 27 nmol/min/mg, which is about ahalf of the activity obtained from GAD
expressed in pYES2-GAD. The expression

Kanbara, M. et al.

69

Fig. 2. Nucleotide and deduced amino acid sequence of GAD1
Nucleotide sequence and deduced amino acid sequence arenumbered on the right. Boxed amino acid residues (Lys318) ismarked as a PLP bind site. Restriction enzyme sites, KpnΙ and SalI,
are shown as underlined.
instead of 69 kDa, which suggests proteolysisof His-tag GAD was not detectable in
may have occurred inside of the yeast cell
SDS-PAGE gel stained with CBB (data not(Fig. 1).
There were no othershown). By using antibody against His-tag,immuno-positive bands observed on theWestern blot analysis gave a band at 15 kDa

70

Expression of yeast glutamate decarboxylase

Fig. 3. Western blot analysis of E. coli transformed with
pET21a-GAD-His6(a) Soluble fraction, induced at 18, 28 and 37 °C. (b)Insoluble fraction, induced at 37 °C. Antibody used was ahorseradish peroxidase-conjugated mouse monoclonal antibody
raised against His-tag.
Western blot analysis. By inspecting theamino acid sequence of GAD deduced fromGAD1 gene sequence, 15 kDa protein portion,
including C-terminal end, should not have
K318, the Lys residue at the active site, toform a Schiff base with PLP (Fig. 2).
Therefore, the activity we have obtained
must come from the remaining portion of
GAD. However, under our experimental
conditions, there was no way to detect it in
the SDS-PAGE by CBB stain. Therefore,
we have concluded that the yeast expression
system should be redesigned or not suitablefor GAD production.
Overexpression of His-tag GAD in
Escherichia coli - An expression systemusing E. coli was constructed for His-tag
GAD.
As described in Materials andMethods, a plasmid pET21a-GAD-His6 wascreated that contained the His-tag element at
the C-terminal of GAD. The cell growth
was carried out at 18°C and 16 h and it gave
GAD activity of ~10 nmol/min/mg for the
crude extract. After the induction carriedout at 37°C with an addition of IPTG, cells,
being harvested by centrifugation anddisrupted by ultrasonication, showed no
GAD activity, probably due to the misfolding.
Under our experimental conditions, E. coli
host cells did not give any residual GAD
activity.
As shown in Fig. 3, while
GAD protein was detected in both soluble
and insoluble fractions, most of GAD
appeared in the insoluble fraction. WhenSDS-PAGE and Western blot analysis werecarriedout,
severalbands
wereKanbara, M. et al.

immuno-positive as observed for the
expression in S. cerevisiae (data not shown),
probably due to the proteolytic cleavage.

71

production of GAD protein. The E. coli
system was found to be more promising than
the yeast system, due to the less proteolysis
that yielded unwanted cleavage on therecombinant GAD. Solubilization of the
inclusion body gave a suitable purity forraising the antibody. We will continue
optimizing the expression system to provide
GAD
suitableforbiochemical
characterization.
ACKNOWLEDGEMENTS
Authors are indebted to Ms.
Yumiko Ueno for her critical reading of themanuscript.

Fig. 4. SDS-PAGE analysis of insoluble
fraction of E. coli.
(a) Insoluble fraction was dissolved in
1% SDS and applied. (b) Dissolved
insoluble fraction was purified on His-tag
column and the purified fraction wasapplied. For His-tag GAD expression, E.
coli strain was grown and the protein
expression was induced at 37 °C for 1 hourin the presence of 1mM IPTG. After
SDS-PAGE, protein bands were visualized
by staining the gels with CBB.
When the major band at 55 kDa wasanalyzed by protein sequencing analysis, thesequence was MKNAGKDASKPN thatmatched to Met-173-Asn184. This suggeststhat N-terminal 172 amino acids were absent
for the 55 kDa protein.
Affinity purification on Ni-column
was carried out for the soluble and insoluble
fractions where the latter was solubilized by
SDS. SDS-PAGE analysis for the insoluble
fraction gave a protein band at around 66
kDa and at 55 kDa (Fig. 4). Amino acidsequencing of 66 kDa band confirmed that itis derived from GAD (data not shown). We
have planned to use the affinity purified
protein for the preparation of an antibody
against GAD.
In this study, both yeast and E. coli
expression systems were attempted for the

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Communicated by Ishijima Sumio