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STUDIA UBB CHEMIA, LXI, 2, Tom I, 2016 (p. 7-19)
(RECOMMENDED CITATION)
EXPRESSION AND PURIFICATION OF RECOMBINANT
PHENYLALANINE 2,3-AMINOMUTASE FROM
PANTOEA AGGLOMERANS
ANDREA VARGAa, ALINA FILIPa, LÁSZLÓ-CSABA BENCZEa,
PÉTER SÁTORHELYIb, EVELIN BELLc, BEÁTA G. VÉRTESSYd,e,
LÁSZLÓ POPPEf,c,*, CSABA PAIZSa,*
ABSTRACT. In the present study the gene of phenylalanine 2,3-aminomutase
from Pantoea agglomerans (PaPAM) was cloned into pET-19b vector and
used for its expression in competent Escherichia coli cells. The recombinant
plasmid, PaPAM-pET-19b, was transformed into competent E. coli strain
BL21(DE3)pLysS cells. Overnight culture of the transformed bacteria was
induced by the addition of isopropylthio-β-D-galactoside (IPTG) to the final
concentrations of 0.1, 0.5 and 1 mM. Also, the effects of different temperatures
(18, 25 and 30°C) and the incubation time of PaPAM were examined. The
fermentation process was scaled up to 10 L fermentor. Affinity purification
conditions were analyzed by SDS-PAGE. The Tm and the activity of the
purified enzyme was also investigated.
Keywords: phenylalanine 2,3-aminomutase, Pantoea agglomerans, optimization,
protein expression
INTRODUCTION
Over the past decades, the preparation of optically pure α-amino
and β-amino acids has received increasing attention because they have
a Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering, Arany János str. 11,
RO-400028, Cluj-Napoca, Romania
b Fermentia Ltd, Berlini u. 47-49., H-1045 Budapest, Hungary
c Department of Organic Chemistry and Technology Budapest University of Technology and
Economics Műegyetem rkp. 3, H-1111 Budapest, Hungary
d Department of Biotechnology and Food Sciences Budapest University of Technology and
Economics Szt. Gellért tér 4, H-1111 Budapest, Hungary
e Institute of Enzymology Research Centre for Natural Sciences of Hungarian Academy of
Sciences Magyar tudósok körútja 2, H-1117 Budapest, Hungary
f SynBiocat Ltd Lázár deák u 4/1, H-1173 Budapest, Hungary
* Corresponding authors: poppe@mail.bme.hu; paizs@chem.ubbcluj.ro
ANDREA VARGA, ALINA FILIP, LÁSZLÓ-CSABA BENCZE, PÉTER SÁTORHELYI, EVELIN BELL,ET AL.
8
many applications in their free form and as fundamental building blocks of
bioactive natural products[1].
An attractive alternative enzymatic route to obtain enantiomerically
pure α- and β-amino acids involves the use of phenylalanine ammonia-lyases
(PALs) and phenylalanine 2,3-aminomutases (PAMs) [2].
Phenylalanine ammonia-lyases and 2,3-aminomutases are emerging
as important enzymatic systems, not only in green synthetic routes to chiral
amines, but also as potential target for treating diseases such as phenylketonuria
and cancer [2].
PAL catalyzes the nonoxidative elimination of ammonia from
L-phenylalanine to give trans-cinnamic acid, whereas PAM catalyzes the
isomerization of α- into β-phenylalanine [3].
PAMs and PALs are members of the class I lyase-like family that
includes tyrosine 2,3-aminomutases (TAMs) [4], tyrosine ammonia-lyases
(TALs)[5], and histidine ammonia-lyases (HALs) [6]. All these enzymes rely
on a protein-derived cofactor, 4-methylideneimidazol-5-one (MIO), which is
generated autocatalytically from three active site residues, Ala-Ser-Gly (Thr-
Ser-Gly in PaPAM), forming a MIO signature motif [7,8].
The reactions catalyzed by these enzymes have considerable potential
for biotechnological applications. They are used as biocatalysts for the synthesis
of (S)-α-amino acids from acrylates (PALs), in kinetic resolution processes for
obtaining (R)-α-amino acids starting from their racemates (PALs), or for the
synthesis of (R)- or (S)-β-amino acids (TcPAM and PaPAM respectively) [9,10,
11,12,13].
Further, although TcPAM and PaPAM are both aryl amino acid 2,3-
aminomutases and share the same cofactor dependency and similarity
(26.7% identity and 42% similarity)[14], they catalyze the α-β-isomerization
with different stereochemistry. TcPAM catalyzes the isomerization of (S)-α-
amino acids to (R)-β-amino acids, whereas PaPAM converts (S)-α-amino
acids to (S)-β-amino acids. In this way, both (R) and (S) enantiomers can
be obtained in enantiomerically pure form.
TcPAM has been widely used as biocatalyst for synthetic procedures
related to β-amino acids but the use of PaPAM as biocatalyst is not so
frequently reported. In the consciousness of this fact, the aim of this work is to
develop methods for recombinant production of phenylalanine 2,3-aminomutase
from Pantoea agglomerans (PaPAM) by optimization and scaling up the
expression process for high level protein expression, in order to obtain PaPAM
efficient biocatalyst for the preparation of enantiomerically pure (S)-β-amino
acids.
EXPRESSION AND PURIFICATION OF RECOMBINANT PHENYLALANINE 2,3-AMINOMUTASE …
9
RESULTS AND DISCUSSION
Optimization of PaPAM overexpression in E. coli
E. coli is still the preferred host for recombinant protein expression
because it is easy to genetically manipulate, it is inexpensive to culture, and
expression occurs fast [15]. pET system also has many advantages that
determine us to use it for the expression of our gene. pET is one of the most
powerful systems developed for the expression of recombinant proteins in E.
coli [16]. E. coli strain BL21(DE3)pLysS is also the most widely used host for
recombinant gene expression.[17] Therefore, E. coli was selected as host for
expression of PaPAM. The aim of this investigation was to examine the effect
of different experimental conditions on the expression of PaPAM in order to
obtain high purity and yields for the enzyme which can be used as biocatalyst.
Inducer concentration optimization
Varying the concentration of IPTG, expression of proteins can be
regulated at different levels, lower level expression can increase the solubility
and activity of the target proteins [18]. With some proteins, it is important to induce
the transcription of the expression plasmid with lower IPTG concentrations, while
others tend to aggregate at high concentrations of IPTG [18].
For the optimization of PaPAM expression, the effect of different
concentrations of inducer (0.1, 0.5 and 1 mM IPTG) was tested on the growing
culture of BL21(DE3)pLysS containing the pET-19b-PaPAM recombinant
plasmid. The SDS-PAGE bands are similar for 0.5 and 1 mM IPTG concentrations
(Figure 1, Lane C and D), indicating the same level of expression of the 72 kDa
PaPAM, despite the increasing inducer concentration. Only a slight difference
can be observed at 0.1 mM IPTG. (Figure 1, Lane B), showing the most
intensive signal at the 72 kDa band of the recombinant PaPAM. Based on these
results, the final concentration of IPTG was set up to 0.1 mM.
Effect of incubation temperature and time on the overexpression of PaPAM
The recombinant plasmid pET-19b-PaPAM was overexpressed to
produce the target protein with an N-terminal His10 tag in E. coli BL21(DE3)pLysS.
Generally, the optimum temperature for the recombinant protein production in
E.coli is 37°C and several studies reported 37°C as the best temperature for
maximum protein production [19]. On the other hand, studies showed that the
rate of expression and culture temperature can affect the proper folding of
recombinant proteins and formation inclusion bodies.[20] Lowering the expression
temperature usually leads to slower growth of bacteria, slower rate of protein
production and lower aggregation of target protein and also most proteases
are less active at lower temperatures [21,22].
ANDREA VARGA, ALINA FILIP, LÁSZLÓ-CSABA BENCZE, PÉTER SÁTORHELYI, EVELIN BELL,ET AL.
10
Figure 1. Induction of the expression of PaPAM by different concentrations of IPTG
in E.coli BL21(DE3)pLysS cells, after 4h. Lane A: protein ladder, Lane B: induction with
0.1 mM IPTG, Lane C: induction with 0.5 mM IPTG, Lane D: induction with 1 mM IPTG,
Lane E: control (0 mM IPTG). The samples were prepared as described in
experimental section.
To evaluate the effect of growth temperature on the expression of
PaPAM after induction, the cultures were incubated at different temperatures
(18, 25, and 37°C). Initially the cell cultures were incubated at 37°C. After the
density of cells reached OD600 ~0.6 (approx. 4 h) the temperature was reduced
(to 18 or 25°C) and the cultures were induced with 0.1 mM IPTG. The density
of the cells was monitored in time (Graphic 1.).
Graphic 1. Effect of growth temperature on the expression of PaPAM
EXPRESSION AND PURIFICATION OF RECOMBINANT PHENYLALANINE 2,3-AMINOMUTASE …
11
Due to the reduced incubation temperature the protein synthesis rate is
slower at 18°C than at 25 or 37°C, longer induction times are necessary for
cells growing. At higher incubation temperature (25 or 37°C), the protein
synthesis is faster and the stationary phase is reached sooner than at lower
temperatures. After 20 h the cells were harvested by centrifugation, followed
by sonication and the protein was purified by metal affinity chromatography on
Ni-NTA resin. The maximum yield of enzyme was obtained in case of 25°C
incubation temperature. The optimal post-induction time on the expression of
PaPAM was 15-16 hours. The determined optimal conditions were also been
used for the large-scale fermentation of E. coli BL21(DE3)pLysS.
Purification using Ni-NTA chromatography
Ni–NTA chromatography system is a rapid and easy purification
technique. Proteins fused with His-tag at either ends (N- or C-terminus)
bind tightly with high affinity on immobilized nickel ions. The strong binding
between His-tag and matrix allows easy washing and efficient elution of
bounded His-tagged protein by competition with imidazole [23].
In the pET-19b vector a His10-tag at the N-terminus is included which is
longer than the usual His6-tag. Lengthening the His-tag increases the affinity of
the enzyme to the Ni-NTA resin. Consequently, higher imidazole concentrations
are required to elute all the bounded enzyme from the resin (from 250 mM up
to 500 mM) [24]. Accordingly, as the SDS-PAGE gel analysis revealed, only
small amount of protein remained in the flow through (Figure 2, Lane D) and in
the washing buffer solutions (Figure 2, Lane E-G).
Figure 2. Purification of PaPAM with Ni-NTA, in absence of protease inhibitor cocktail.
Lane A: protein ladder, Lane B: supernatant, Lane C:pellet, Lane D: flow through
Lane E: LS1, Lane F: HS, Lane G:LS2, Lane H: 20 mM Imidazole, Lane I:
500 mM Imidazole, Lane J: 1 mM Imidazole. The samples were prepared
as described in experimental section.
ANDREA VARGA, ALINA FILIP, LÁSZLÓ-CSABA BENCZE, PÉTER SÁTORHELYI, EVELIN BELL,ET AL.
12
It has been observed that PaPAM activity descreased after elution
from the Ni-NTA column probably due to prolonged exposure to very high
imidazole concentration and we also observed that the obtained enzyme
has some unspecific bands on the SDS-PAGE gel (Figure 2, Lane I) due to
the protease activity for the enzyme. In order to eliminate these facts we
added protease inhibitor cocktail to the lysis buffer and we reduced de
imidazole concentration, testing 250, 350, 450, 500 mM imidazole solutions
for protein elution. The best result was obtained by elution with 350 mM
imidazole, resulting in protein solution appearing as single band on the
SDS-PAGE (Figure 3, Lane I), indicating a highly purified enzyme.
Thermal stability
Thermal shift (ThermoFluor) assays offer a rapid and simple technique
for assessing the thermal stability of proteins and to investigate factors
affecting this stability. An environmentally sensitive fluorescent dye is used to
monitor protein unfolding with respect to temperature [25]. In the ideal case,
no fluorescence is observed at low temperature because the protein is
completely and correctly folded and no hydrophobic areas are exposed.
Upon an increase in temperature the protein starts to unfold and hydrophobic
areas become exposed and the fluorescent dye can bind to these areas and
fluorescence occurs.
Figure 3. Purification of PaPAM with Ni-NTA, in presence of protease inhibitor cocktail.
Lane A: protein ladder, Lane B: supernatant, Lane C: flow through, Lane D: pellet, Lane E:
LS1, Lane F: HS, Lane G:LS2, Lane H: 20 mM Imidazole, Lane I: 350 mM Imidazole, Lane
J: 1 mM Imidazole. The samples were prepared as described in experimental section.
The PaPAM enzyme presents a good thermal stability, it can be observed
in Figure 4 that its maximum melting temperature is approximately 73C.
EXPRESSION AND PURIFICATION OF RECOMBINANT PHENYLALANINE 2,3-AMINOMUTASE …
13
Figure 4. The first derivative of the melt curves.
We also investigated if the substrate of the enzyme has a stabilizing
effect. Therefore following measurements were performed in TRIS buffer
solution, pH 8.5 in the presence of L-phenylalanine.
The melting temperature was read from the negative curve of the
first derivative of the experimental curve. By comparing the results of the
measurements obtained in presence and in absence of the substrate, it can
be observed that the modification of melting temperature in presence of L-
phenylalanine is minor (from 73C to 74C), suggesting no stability increase
of the enzyme upon substrate binding.
Activity assay
In nature, phenylalanine 2,3-aminonutase from Pantoea agglomerans is an
(S)-selective enzyme, transforming the (S)-α-phenylalanine in (S)-β-phenylalanine [8].
The activity and the selectivity of PaPAM was tested using rac-α-
phenylalanine as substrate (Scheme 1). HPLC analysis of the reaction supports
the formations of the (S)-β-phenylalanine (Figure 5B) with maximum selectivity
(compared to the racemic mixture, Figure 5A).
Scheme 1. Transformation of the rac-α-phenylalanine by PaPAM.
ANDREA VARGA, ALINA FILIP, LÁSZLÓ-CSABA BENCZE, PÉTER SÁTORHELYI, EVELIN BELL,ET AL.
14
Figure 5. HPLC chromatograms for: A. rac-β-phenylalanine as control.
B. Transformation of rac-α-phenylalanine by PaPAM.
CONCLUSIONS
We examined different experimental conditions regarding the expression
and purification of PaPAM in order to obtain high purity and yield for the enzyme.
IPTG concentrations, various post-induction temperatures on the expression,
the imidazole concentration in the purification steps, the thermal stability and
the activity of the enzyme were also examined.
The results showed that induction with 0.1 M IPTG was sufficient to
induce the expression of PaPAM, which is 10 times less than normally used
IPTG concentration. Moreover, 15 hour post-induction incubation at 25°C was
found to be optimal for producing a higher level of PaPAM. Reducing the
imidazole concentration to 350 mM and adding protease inhibitor cocktail
improved the stability of the yielding enzyme. Furthermore, electrophoretically
pure recombinant PaPAM enzyme preparation was obtained using Ni affinity
chromatography.
The detemined optimized parameters were also successfully applied
for large scale fermentation of PaPAM, obtaining high levels of protein.
EXPERIMENTAL SECTION
PaPAM gene synthesis and cloning
The gene of the Pantoea agglomerans PAM (Uniprot code: Q84FL5,
PBD code: 3UNV, encoding 623 AA – Figure 6) was optimized to the codone
usage of E. coli. The 1639 bps long synthetic gene was produced by Life
A.
B.
EXPRESSION AND PURIFICATION OF RECOMBINANT PHENYLALANINE 2,3-AMINOMUTASE …
15
Tehnologies. The gene was cloned into pET-19b host vector using XhoI and
Bpu1102I cloning site (Figure 7). The pET-19b vector in which the PaPAM
gene was cloned contains a His10-tag N-terminal sequence, an enterokinase
cleavage site, a gene resistant to ampicillin and the T7lac promoter sequence.
In the supplementation of the growth medium ampicillin was replaced by
carbenicillin, due to the highest stability of later.
wt-PaPAM
MSIVNESGSQPVVSRDETLSQIERTSFHISSGKDISLEEIARAARDHQPV
TLHDEVVNRVTRSRSILESMVSDERVIYGVNTSMGGFVNYIVPIAKASEL
QNNLINAVATNVGKYFDDTTVRATMLARIVSLSRGNSAISIVNFKKLIEI
YNQGIVPCIPEKGSLGTSGDLGPLAAIALVCTGQWKARYQGEQMSGAMAL
EKAGISPMELSFKEGLALINGTSAMVGLGVLLYDEVKRLFDTYLTVTSLS
IEGLHGKTKPFEPAVHRMKPHQGQLEVATTIWETLADSSLAVNEHEVEKL
IAEEMDGLVKASNHQIEDAYSIRCTPQILGPVADTLKNIKQTLTNELNSS
NDNPLIDQTTEEVFHNGHFHGQYVSMAMDHLNIALVTMMNLANRRIDRFM
DKSNSNGLPPFLCAENAGLRLGLMGGQFMTASITAESRASCMPMSIQSLS
TTGDFQDIVSFGLVAARRVREQLKNLKYVFSFELLCACQAVDIRGTAGLS
KRTRALYDKTRTLVPYLEEDKTISDYIESIAQTVLTKNSDI
Figure 6. Amino acids sequence of recombinant wt-PaPAM.
Figure 7. pET-19b-PaPAM vector map.
The vector map was generated using Snapgene.
Transformation in E.coli host cells
Transformation of plasmid DNA into E.coli XL1-Blue (for plasmid
amplification) and BL21(DE3)pLysS (for expression) was performed using
the heat shock method, inserting the foreign plasmid into bacteria. After 20 min.
ANDREA VARGA, ALINA FILIP, LÁSZLÓ-CSABA BENCZE, PÉTER SÁTORHELYI, EVELIN BELL,ET AL.
16
incubation on ice, the mixture of 100 µL chemically competent bacterial cells
and 2 µL of plasmid DNA was incubated at 42°C for 45 seconds (heat shock)
and then placed back on ice for 25 min. 200 µL SOC media was added and
the transformed cells were incubated at 37°C for 1 h. In case of XL1-Blue
transformation the transformed bacteria were plated on LB agar-plates
containing tetracycline (30 μg/mL) and carbenicilin (50 μg/mL). In case of
BL21(DE3)pLysS transformation 50 µg/mL carbenicillin and 30 µg/mL
chloramphenicol were used. Both cultures were then incubated overnight at
37°C, forming single colonies of bacteria bearing the plasmid encoding the
recombinant protein.
Expression and production of the recombinant PaPAM
The recombinant PaPAM carrying N-terminal (His)10-tag was overexpressed
in E.coli host cells (BL21(DE3)pLysS. For the expression step, a colony of the
transformed plasmid was grown overnight at 37°C in 5 mL of Luria-Bertani (LB)
medium containing carbenicillin (50 µg/mL) and chloramphenicol (30 µg/mL).
A 0.5 L of LB medium was inoculated with 1% (v/v) of the overnight culture in
an Erlenmeyer flask and grown until the measured optical density at 600 nm
(OD600) reached 0.6-0.7 at 37°C (optimal temperature for E. coli growth) and
only at the induction phase the temperature was reduced to 18, 25, 37°C and
the cells were induced by the addition of (0.1, 0.5, 1 mM) IPTG. The culture was
shaken at 200 rpm for 16 h.
Large scale fermentation of the recombinant PaPAM
Ten litres of Luria-Bertani (LB) medium were sterilized at 121°C, 1.2
bar for 25 min. After sterilization, the media was cooled down to 37°C and
ampicillin sodium salt was added to the fermentation broth for a final
concentration of 100 µg/mL. The fermentation media was inoculated with 100
mL of the overnight seed culture of the E. coli producer strain. The following
fermentation parameters were set up: temperature at 37°C, agitation 300 rpm,
overpressure 0.2 bar and bottom air inlet 5 L/min. The pH value of the
fermentation broth was controlled at pH 7.1±0.1. The dissolved oxygen (DO)
value was set to a minimum of 30% and was controlled by the stirring speed.
When the OD600 of the culture reached 0.7±0.1, the temperature was set to
25°C and the culture was induced with IPTG (0.1 mM final concentration),
The fermentation broth was harvested after 16 h when the culture reached
the stationary growth phase.
Purification of the recombinant PaPAM
The cells were harvested by centrifugation (25 min, 5000×g) and
resuspended in 50 mL lysis buffer (150 mM NaCl, 50 mM TRIS pH 7.5,)
EXPRESSION AND PURIFICATION OF RECOMBINANT PHENYLALANINE 2,3-AMINOMUTASE …
17
supplemented with DNAse, RNAse, Lysosyme, 2 mM PMSF and EDTA-
free protease-inhibitor cocktail. Further, the cells were lysed by sonication
and cell debris was removed by centrifugation (10000 × g, 30 min).
The proteins were further purified using a Ni-NTA-agarose column
and washed with different buffer solutions.
The column was washed: LS (low salt) buffer, pH 7.5 (4-5V; V=resin
volume), HS (high salt) buffer, pH 7.5 (2V), LS (2-4V), LS with 20 mM
imidazole, LS with (250, 350, 450, 500 mM) imidazole (protein elution), LS
with 1 M imidazole (4V), ddH2O (7V) and 20% ethanol (1-2V), the resin was
stored in 20% ethanol at 4°C.
The resulting eluate was dialyzed in 50 mM PBS, 4-5h at 4°C. The
purity of the resulting fractions was verified by SDS-PAGE analysis. The
samples were boiled for 5 min in Laemmli buffer and were loaded on a 12%
SDS-PAGE. After dialysis the fractions containing purified protein were
concentrated by centrifugal ultrafiltration. The concentration of the purified
protein was determined by Bradford method.
Optimization parameters
To determine the optimal IPTG concentration on the recombinant
protein production, the recombinant clone culture was grown to OD600 of about
0.6-0.7, and induced by adding IPTG to final concentrations of 0.1, 0.5 and 1
mM. After 4 hours of induction, 1 mL samples were colected, centrifuged and
the pellet resuspended in 0.5 mL dH2O. The samples were lysed by boiled for
5 min in Laemmli buffer, and were loaded on a 12% SDS-PAGE.
To examine the effect of temperature, recombinant protein expression
was induced by addition of 0.1 mM IPTG at different temperatures (18, 25 and
37°C). The OD600 was monitored by time, after 20 hours of induction, cells
were harvested by centrifugation, followed by sonication and the protein was
purified by Ni-NTA.
Thermostability assay
The thermal stability of the enzyme was determined by thermofluor
measurements using real-time PCR as described in the literature.[26]
The assay is performed using a 96-well iCycler iQ Real Time PCR
plate. The total reaction volume is 25 µL and the plate is set up on ice. 20
µL of 0.1M TRIS buffer solution is pipetted into the wells of the plate. A
5000 × SYPRO Orange stock solution in DMSO is diluted 1:100 in water
and 1 µL is added to the 20 µL well solution which contains 1.5 mg/mL
PaPAM protein. The plates are sealed with Optical Quality Sealing Tape
and centrifuged at 2000 rpm for 1 min. The plate is heated from 20 to 90°C
in 1°C increments in of iCycler iQ Real Time PCR Detection System.
ANDREA VARGA, ALINA FILIP, LÁSZLÓ-CSABA BENCZE, PÉTER SÁTORHELYI, EVELIN BELL,ET AL.
18
Activity assay
PaPAM activity was determined by the conversion of (S)-α-Phe to
(S)-β-Phe. Into the solution of rac-α-phenylalanine (2 mg) in TRIS buffer
(100 mM, pH 8.0, 2 mL), wt-PaPAM (0.8 mg) was added and the reaction
mixtures were stirred at room temperature. A 100 µL sample was taken and
the reaction was stopped by heating for 10 min at 90°C in the presence of a
small amount of activated charcoal. After filtration, the sample was
analyzed with high performance liquid chromatography (HPLC), using a
Chiralpak ZWIX(+) column, and MeOH (50 mM formic acid and 100 mM
diethyl amine):ACN:H2O 49:49:2 (v/v/v) as eluent, respectively.
ACKNOWLEDGMENTS
AV thanks for the financial support of the Sectorial Operational
Programme for Human Resources Development 2007-2013, co-financed by
the European Social Fund, under the project POSDRU/159/1.5/S/137750 -
“Doctoral and postdoctoral programs - support for increasing research
competitiveness in the field of exact Sciences”. CP thanks for financial support
from the Romanian National Authority for Scientific Research, CNCS –
UEFISCDI (PN-II-IDPCE-2011-3-0799). This work was also supported by the
Hungarian OTKA Foundation (Project NN-103242), by the New Hungary
Development Plan (Project TÁMOP-4.2.2.B-10/1–2010–0009), by the
Hungarian Research and Technology Innovation Fund (KMR 12-1-2012-0140)
and by the EU COST (Action CM1303 “SysBiocat”).
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