Beruflich Dokumente
Kultur Dokumente
14 August 2014
Protein Expression and Purication xxx (2014) xxxxxx
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Jorge Gonzlez-Bacerio a,, Joel Osuna b, Amaia Ponce a, Rafael Fando c, Katherine Figarella d,
Yanira Mndez a, Jean-Louis Charli b, Mara de los . Chvez a
a
Centro de Estudio de Protenas, 25 # 455 entre J e I, Facultad de Biologa, Universidad de La Habana, Cuba
Instituto de Biotecnologa, Universidad Nacional Autnoma de Mxico (UNAM), Ave. Universidad 2001, Cuernavaca, Mor., Mexico
Centro Nacional de Investigaciones Cientcas, Ave. 25 y 158, 12100 La Habana, Cuba
d
Fundacin Instituto de Estudios Avanzados, Carretera Nacional de Hoyo de la Puerta, Valle de Sartenejas, Baruta 1080, Estado Miranda, Venezuela
b
c
a r t i c l e
i n f o
Article history:
Received 8 July 2014
and in revised form 3 August 2014
Available online xxxx
Keywords:
Expression in Escherichia coli
IMAC
M1-family aminopeptidase
Malaria
PfA-M1
Plasmodium falciparum
a b s t r a c t
Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo
proteases, is a promising target for malaria, a devastating human parasitic disease. We report the highlevel expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and
an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector.
Optimal expression was achieved by induction with 1 mM IPTG at 37 C for 18 h. This allowed obtaining
100 mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass
was from rPFAM1. rPfAM1, fused to an amino-terminal 6His tag, was puried in a single step by immobilized metal ion afnity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral
optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition prole typical of metallo-aminopeptidases, and inhibition from Zn2+ excess, were similar to those
of the native PfAM1. We have thus optimized an expression system that should be useful for identifying
new PfAM1 inhibitors.
2014 Published by Elsevier Inc.
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Introduction
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Abbreviations used: AP(s), aminopeptidase(s); PfAM1, M1-aminopeptidase from P.
falciparum; p96, PfAM1 96-kDa form; p68, PfAM1 68-kDa form; ePepN, M1aminopeptidase from E. coli; rPfAM1, recombinant PfAM1; IMAC, immobilized metal
ion afnity chromatography; DNA, deoxyribonucleic acid; G, guanine; C, cytosine;
PCR, polymerase chain reaction; LB, Luria-Bertani; OD600(280)nm, optical density at 600
or 280 nm; IPTG, isopropyl-b-D-1-thiogalactopyranoside; SDSPAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; EA, enzymatic activity; Leu, leucine; pNA,
p-nitroanilide; C1, culture of E. coli BL21 non-transformed and induced; C2, culture of
E. coli BL21 transformed with the pTrcHis2B vector and induced; C3, culture of E. coli
BL21 transformed with the pTrcHis2B-rPfAM1 construct and non-induced; BSA,
bovine serum albumin; LCMS/MS, liquid chromatography coupled to tandem mass
spectrometry; UNAM, Autonomous National University of Mexico; His, histidine;
DMSO, dimethyl sulfoxide; U, unit of EA; MES, 2-(N-morpholino)ethanesulfonic acid;
Arg, arginine; Lys, lysine; Ala, alanine; Val, valine; Ile, isoleucine; Gly, glycine; Pro,
proline; bp, base pairs; CAI, Codon Adaptation Index; spEA, specic EA; AMC, 7amido-4-methylcoumarin; Glu, glutamic acid; bNA, b-naphtylamide; dNTPs, deoxynucleoside-triphosphates; PMSF, phenyl-methylsulfonyl-uoride.
http://dx.doi.org/10.1016/j.pep.2014.08.002
1046-5928/ 2014 Published by Elsevier Inc.
Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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The services of the company GeneArt AG (Germany) were contracted. The rpfam1 gene (protein sequence available in UniProtKB/
TrEMBL: Q8IEK1) was optimized for its expression in bacteria,
using the software GeneOptimizer. The optimization consisted
of changing the codons of the native gene to codons with the
highest usage in bacteria, increasing the G/C content and avoiding
the formation of sequences that can affect transcription and
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falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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without lactose and with 0.26% glucose). The nal OD600 nm was
measured, and expression was evaluated as previously mentioned.
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rPfAM1 purication
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solubilized in DMSO. The absorbance at 405 nm, due to the liberation of pNA, was monitored at 15-s intervals for 3 min. Kinetic
assays were conducted with volumes of protein extracts or concentrations of puried rPfAM1 that were linearly related to the initial
rates, at 37 C, in 96-well plates (200 lL nal volume) using a
microplate spectrophotometer (Multiscan FC, Thermo Scientic,
USA). The assays conditions were 50 mM TrisHCl buffer, pH
7.27.4. The nal DMSO concentration was not higher than 2%.
The used molar extinction coefcient at 405 nm for pNA was
9.87 mL lmol1 cm1 [31]. The EA unit (U) is the amount of
enzyme that hydrolyzes 1 lmol of substrate per minute under
the assays conditions. The assays were performed in triplicate,
and the results are presented as the mean standard deviation.
The inhibition of puried rPfAM1 by 1 mM 1,10-phenanthroline
(Sigma, USA), solubilized in DMSO, was tested pre-incubating the
enzyme-inhibitor mixture for 5 min at 25 C before the addition
of substrate. The control was prepared by pre-incubating the
enzyme with the same volume of DMSO. Residual activity was
dened as the ratio between the EA in presence of the inhibitor
and the activity of the control. The effect of the Zn2+ concentration
on the EA of rPfAM1 was evaluated with 0, 10 or 100 mM ZnCl2
(Sigma, USA) and 1.5 mM Leu-pNA substrate. To test the effect of
pH, the reaction mixtures were adjusted to different pH values with
100 mM MES (pH 5.2, 5.5 and 6.5) or 50 mM TrisHCl (pH 7.2, 7.5,
8.0, 8.5 and 9.0). To test substrate specicity, aminoacyl-pNA
chromogenic substrates (Arg-, Lys-, Leu-, Ala-, Val-, Ile-, Gly- and
Pro-pNA) (Bachem, Sweden) were tested at different concentrations
(range: 15.6500 lM) at pH 7.27.4 (50 mM TrisHCl buffer), and
the kinetic parameters KM and kcat were calculated from LineweaverBurk plots. The initial enzyme concentration was calculated
assuming a sequence-based predicted molecular weight for rPfAM1
of 106.13 kDa, determined using the Sequence Manipulation Suite v
2.0 software, (http://www.bioinformatics.org/sms2/).
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Protein analysis
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AP EA assays
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Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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Fig. 2. Optimization of the rpfam1 gene for expression in E. coli. Data for the native pfam1 gene (without the region encoding the amino-terminal extension) and the synthetic
rpfam1 gene. Distribution of codons according to quality ranges (A). A value of 100 is set for the codon with the highest usage frequency for a given amino acid in E. coli [32].
Distribution of the quality of codons (B) and of the G/C content (C) along the sequence of the native and synthetic genes. Plots in (C) show the G/C content in a 40-bp window
centered at the indicated nucleotide position. For sequence analysis and graphic visualization, we used the web site GeneScript (http://www.genscript.com/cgi-bin/tools/
rare_codon_analysis).
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Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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Fig. 3. Construction of plasmid pTrcHis2B-rPfAM1. (A) Cloning of the rpfam1 gene in the pTrcHis2B vector. The lacIq gene and the pBR322 replication origin were omitted in
the pTrcHis2B-rPfAM1 scheme for simplicity. (B) Four clones of the genetic construct pTrcHis2B-rPfAM1 were checked by electrophoresis on 1% agarose gel.
Fig. 4. Preliminary expression of rPfAM1 in E. coli BL21. Expression at a small scale (5 mL), in LB broth supplemented with ampicillin at 37 C; induction for 4 h with 1 mM
IPTG added at the exponential phase of growth. Negative controls of expression: non-transformed strain and induced, strain transformed with pTrcHis2B vector and induced,
and strain transformed with pTrcHis2B-rPfAM1 construct and not induced. Evaluation of expression by SDSPAGE (8% acrylamide, non-reducing conditions, Coomassie
staining) (A) and determination of AP spEA (Leu-pNA substrate) in cell-free soluble protein extracts (B). Lanes contained a constant amount of supernatant (20 lg) or of the
pellet protein. MWM: molecular weights markers. Data in (B) are the mean standard deviations (n = 3). (+): presence. (): absence. (C) Sequence of tryptic peptides (by LC
MS/MS) derived from the 100-kDa protein band (enclosed by an oval). Identied peptides that match with the PfAM1 sequence (51% of sequence coverage) are underlined.
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falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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Fig. 5. Comparative analysis of three induction methods for rPfAM1 expression in E. coli BL21. Evaluation of expression by SDSPAGE (8% acrylamide, non-reducing
conditions, Coomassie staining) after 18 h-induction with 0.1 mM IPTG added at the beginning of the culture in ampicillin-supplemented LB broth (A), 1 mM IPTG added at
late exponential phase of bacterial growth in ampicillin-supplemented LB broth (B), or auto-induction with 0.2% of lactose in ampicillin-supplemented ZYMB-5052 culture
media (C). Negative controls of expression are as presented in Fig. 4. Lanes contain a constant amount of supernatant (20 lg) or of pellet protein. rPfAM1 bands are enclosed
by ovals. (D) Evaluation of expression by determination of AP spEA (Leu-pNA substrate) in cell-free soluble protein extracts. The means standard deviations (n = 3). (+):
Presence. (): absence.
Table 1
Effect of three induction methods on rPfAM1 expression in E. coli BL21.
Induction method
0.1 mM IPTG
1 mM IPTG
Auto-induction
(0.2% lactose)
Final
OD600
3.6
3.9
3.7
nm
Approximate level of
expression
(% of total proteins)a
Insoluble fraction
Soluble fraction
2.0 0.3
4.1 0.2
3.6 0.9
9.4 2.2
19 0.8
11 1.2
Approximate volumetric
yield (mg of soluble rPfAM1/
L of culture)a
spEA
(104 U/mg of protein)
Expression level:
fold increment
over basal spEA
Total EA (U)
61 14
102 4
86 9
192 12
220 10
145 7
11 1b
10 2b
4 0.2c
1.25 0.08
1.17 0.05
1.15 0.06
Means standard deviations (n = 3). OD600 nm: optical density at 600 nm. EA: enzymatic activity. spEA: specic EA. U: unit of EA, dened as the amount of enzyme hydrolyzing
1 lmol of substrate per min under the assays conditions.
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Determined by gel densitometry with ImageJ 1.38d software.
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The mean activities registered in negative controls of expression (non-transformed strain, strain transformed with pTrcHis2B vector and strain transformed with genetic
construct pTrcHis2B-rPfAM1 but without induction) were taken as the basal activity.
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In this case, we considered as basal activity the activity measured in the non-induced control.
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Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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protein, a critical parameter to the success of the purication process, was obtained with 1 mM IPTG induction. Therefore, this
method was selected for routine production of rPfAM1.
Selection of induction time and number of ultrasonic cycles for
extraction of recombinant protein
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A growth curve, representative of the E. coli BL21/pTrcHis2BrPfAM1 strain, is shown in Fig. A.2.A. The stationary phase was
reached 12 h after induction. The specic AP activity was
progressively augmented, with the highest value detected at 18 h
post-induction, which was selected for the production of the
recombinant enzyme. On the other hand, two ultrasonic cycles
were sufcient for the maximal recovery of total proteins and AP
specic activity, and spEA was notably reduced with four cycles
(Fig. A.2.B). Two cycles of ultrasonic treatment were selected for
rPfAM1 extraction from E. coli BL21/pTrcHis2B-rPfAM1 cells.
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Purication of rPfAM1
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Purication of rPfAM1 in a single step from an E. coli BL21 protein extract enriched in the recombinant enzyme was performed
by IMAC. The chromatographic prole reveals that rPfAM1 elution
occurred at imidazole concentrations as low as 20 and 30 mM
(Fig. 6A). SDSPAGE indicated that these chromatographic fractions contained a 100-kDa protein that was highly concentrated
and accompanied by other proteins. The main additional bands
had molecular masses approximately 50 and 200 kDa. Then,
50 mM imidazole was selected for elution, as this concentration
value was sufcient to achieve total desorption of the recombinant
protein. The low afnity of rPfAM1 to the matrix in a native IMAC
could be explained by an incomplete exposure of the 6His tag in
the protein surface, and/or the use of a cobalt resin, which releases
histidine-tagged proteins with gentler conditions regarding nickel
resins (www.thermosher.com).
In Fig. 6B, a representative IMAC of the rPfAM1-enriched protein extract is shown. A 280-nm absorbance peak, associated with
the AP EA, was obtained. In contrast, if chromatography was
applied to a negative control extract (E. coli BL21/pTrcHis2B culture), this absorbance peak was not detected. The evaluation of
chromatographic runs by SDSPAGE and Coomassie staining indicated that if the negative control extract was loaded onto the column, elution with 50 mM imidazole did not lead to detection of
any band; on the contrary, if the IMAC column was loaded with
the extract from E. coli BL21/pTrcHis2B-rPfAM1-H6N, we observed
one highly concentrated 100 kDa protein in the eluates in addition to minor bands, including the 200- and 50-kDa bands.
Fractions 1 and 2 from the eluates of various chromatographic runs
loaded with the rPfAM1-enriched extract were pooled and dialtered with a 10-kDa cut-off membrane. Dialtration was performed to concentrate the recombinant protein and to remove
imidazole, which interferes with the EA assays for metallo-APs.
We sequenced tryptic peptides obtained from the 100- and
50-kDa protein bands by LCMS/MS (Fig. 7). We corroborated
that both bands corresponded to rPfAM1, with 24 sequenced peptides in the rst band (55% of sequence coverage) and 8 peptides
for the second band (23% of sequence coverage). The position of
the identied peptides in the PfAM1 sequence from the 50-kDa
band indicates that this protein is an amino-terminal degradation
product of rPfAM1. The identication of this amino-terminal degradation product is in agreement with the report of McGowan
et al. [20] about the purication of another recombinant variant
of PfAM1. These authors obtained an amino-terminal degradation
product with a molecular mass of 55 kDa, the presence of which
did not hamper the successful determination of the PfAM1
three-dimensional structure by X-ray crystallography with a
2.1- resolution. The selective adsorption of an amino-terminal
degradation product to the IMAC column (Fig. 7) is consistent with
the presence of a histidine tag at the carboxyl terminus of rPfAM1
that is properly exposed in the cleaved molecule for its interaction
with the Co2+ resin.
To test whether other protein bands coeluting with the
100-kDa band were additional rPfAM1 degradation products, a
Western blot with a commercial anti-6His tag antibody was performed on the eluates from IMACs loaded with extracts derived
from the E. coli BL21/pTrcHis2B and E. coli BL21/pTrcHis2BrPfAM1-H6N cultures (Fig. 8). The anti-His antibody did not interact with host proteins but recognized the 100-kDa band rPfAM1
and additional bands in the dialtered fraction, including that of
200-kDa. This indicates that the minority bands which elute
together with rPfAM1 on IMAC are degradation products of the
recombinant protein. Furthermore, as the antibody recognized several components in the rPfAM1-enriched extract, degradation
occurred before protein extract application to the column. The
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Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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Fig. 6. rPfAM1 purication by IMAC. (A) Determination of the imidazole concentration that allows elution of rPfAM1 adsorbed to a Co2+ matrix. (B) Representative IMAC for
rPfAM1 purication. A comparison with the extract that does not contain rPFAM1 (derived from an E. coli BL21/pTrcHis2B culture) is shown. The chromatographic runs were
evaluated by SDSPAGE (8% of acrylamide, reducing conditions, Coomassie staining). Protein samples applied on gels were normalized by volume. MWM: molecular weight
markers. Ext: rPfAM1-enriched protein extract. FT: ow-through sample of the column. EA: enzymatic activity. El: eluate.
Fig. 7. Identication of the 100-kDa (triangle) and 50-kDa (arrow) bands obtained as a result of rPfAM1 purication by IMAC. The identication was performed by the
sequencing of tryptic peptides derived from the gel bands (8% of acrylamide, reducing conditions, Coomassie staining) by LCMS/MS. The amount of protein applied in the gel
was normalized by volume. Ext: rPfAM1-enriched protein extract. FT: ow-through sample of the column. IMAC: pool of eluates from various IMAC runs. Dialt: dialtered
rPfAM1 with a 10-kDa cut-off membrane. M: molecular weight markers.
Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
YPREP 4545
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Fig. 8. Western-blot analysis of the expression and purication of rPfAM1 with an anti-histidine antibody. SDSPAGE (8% of acrylamide, reducing conditions); the gel was
stained with Coomassie (left); proteins were transferred to a nitrocellulose membrane and revealed with an anti-His antibody (right). The amount of protein applied to the gel
was normalized by volume. Ext: protein extract. FT: ow-through sample. El: eluate. M: molecular weight markers. Diaf: dialtered rPfAM1 with a 10-kDa cut-off membrane.
Table 2
Summary of the rPfAM1 purication process.
Purication step
EA (U)
Yield (%)
spEA (U/mg)
Purication
factor (fold)
Extract
IMAC
Dialtration
72.5 3.7
4.57 0.34
17.2 1.16
100
6.3
24
0.56 0.03
0.73 0.05
15.3 1.03
1
1.3
27.3
487
492
The Zn2+ divalent cation is present in the active site of metalloAPs belonging to the M1 family [35], where it acts as an enzymatic
cofactor [36]. The presence of Zn2+ in the active site of rPfAM1 is
suggested by the 80% inhibition caused by 1 mM 1,10-phenanthroline, a metal-ion chelator. Fig. 9A shows that the AP activity of the
puried recombinant enzyme was diminished by 50% with 10 lM
ZnCl2 and by more than 80% when the concentration was raised to
100 lM. A qualitatively similar result was obtained with ZnSO4
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Fig. 9. Partial kinetic characterization of rPfAM1. (A) Effect of the Zn2+ concentration (ZnCl2) on the rPfAM1 enzymatic activity. Substrate: 1.5 mM Leu-pNA. Buffer: 50 mM
TrisHCl, pH 7.27.4. Means (n = 3). (B) Effect of pH on the rPfAM1 EA. Substrate: 1.5 mM Leu-pNA. Buffers: 100 mM MES (pH 5.2, 5.5 and 6.5; triangles) and 50 mM TrisHCl
(pH 7.2, 7.5, 8.0, 8.5 and 9.0; squares). Means (n = 3). (C) Effect of substrates on rPfAM1 EA; substrate concentration range: 15.625500 lM. Buffer: 50 mM TrisHCl, pH 7.2
7.4. The kinetic parameters for each substrate were calculated from LineweaverBurk plots. The substrates with the highest specicity constants (kcat/KM) are highlighted in
bold. kcat: catalytic constant or turnover number. ND: Not determined.
Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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(data not shown), indicating that the inhibitory effect is not caused
by the anion. This suggests that the Zn2+ associated to the active
sites is not lost during purication and indicates the uselessness
of supplementing the enzymatic preparation with this cation. A
similar result was reported for the neutral AP from Bombyx mori,
which is activated at very low Zn2+ concentrations but inhibited
with ion concentrations higher than 100 lM [37].
The maximal rPfAM1 activity toward the Leu-pNA substrate was
registered in the pH range 7.27.4, with an abrupt fall between pH
8.0 and 9.0 (Fig. 9B). The reduction of the activity in the acid zone
was more gradual, as indicated by the detection of 40% activity at
pH 5.25.5 compared with the maximal value. For native PfAM1,
AP activity toward Leu-AMC is optimum at pH 7.4, but at least
40% of activity remains between pH 5.8 and pH 8.6 [9]. As some
authors afrm that PfAM1 performs its biological function in the
lumen of the parasite acidic digestive vacuole [26], where pH values
range from 5.0 to 5.5 [38], attention has been focused on enzyme
activity in acidic conditions. Likewise, mammalian M1-family basic
AP acts in acidic vesicles and shows a high activity at pH 5.0 [39].
However, another group reported that native and recombinant
PfAM1 have an optimum pH of 7.0 toward Arg-AMC, with an activity <20% at pH 6.0 and a total loss of activity at pH 5.0 [20]. The discrepancy in remaining activity at pH < 6.0 might be due to
dissimilar enzyme concentrations or buffers in each pH range, as
enzyme activity can vary according to the buffer identity at a xed
pH and enzyme concentration [40]. Unfortunately, this information
is not always available. In correspondence with this idea, we
observed that the activity of rPfAM1 toward Leu-pNA at pH 5.2
reached 40% of maximal activity (obtained at pH 7.27.4) when
it was measured in a MES buffer, but it was only 10% of the maximal activity when an acetate buffer was used (data not shown).
This is the rst report of the kinetic parameters of PfAM1 determined with aminoacyl-pNA-conjugated substrates. The higher values of the specicity constant were obtained with the substrates
Lys-, Arg-, Leu- and Ala-pNA, in that order (Fig. 9C). It was not possible to determine the kinetic parameters with the isoleucine-,
valine-, glycine- and proline-based substrates because activity
was not detected, even at the maximal substrate concentration
used of 500 lM. The preference order of rPfAM1 for substrates is
in agreement in general terms with what has been previously
reported. With a single dose of aminoacyl uorogenic substrates
and neutral pH, the order of preference of native PfAM1 for the ve
best P1 residues was lysine > alanine > arginine > leucine > phenylalanine [9]. Two similar studies with the same rPfAM1 form that
determined the specicity constants toward uorogenic substrates
demonstrated a preference order for P1 amino acids of (methionine = leucine) > (alanine = arginine) > tryptophan > phenylalanine; the specicity constant toward lysine in the P1 position of
the substrate was not reported [20,41].
The substrate specicity of another variant of PfAM1 determined through the specicity constant toward the dipeptides XAla revealed the following order of preference for the seven best
P1 residues: lysine > methionine > arginine > (leucine = phenylalanine) > tyrosine > alanine [42]. The preference for lysine in the P1
position is in agreement with the results obtained in the present
work (Fig. 9C) as well as with what has been reported for native
PfAM1 toward aminoacyl-AMC conjugates [9]. The position of
methionine in the order of preference agrees with the data generated with uorogenic substrates [20,41]. The assignation of arginine and leucine to the third and fourth positions in the
hierarchy of PfAM1 specicity [42] is also consistent with our data
(Fig. 9C) and previous data. However, the relatively poor hydrolysis
of the dipeptide Ala-Ala does not match the specicity preference
toward the Ala-containing uorogenic conjugates, most likely due
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Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002
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Acknowledgments
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This work was supported by Grant F/4730-1 from the International Foundation for Science (IFS, Sweden) (2010-2012), fellowship 811.06.03 from Secretara de Relaciones Exteriores del
Gobierno de Mxico (2010), and fellowship from Universidad de
las Naciones Unidas, Programa de Biotecnologa para Latinoamrica y el Caribe (UNU-BIOLAC) (2012) to J.G.B.; Grant J000.0453 from
Consejo Nacional de Ciencia y Tecnologa (CONACYT, Mexico)
Ministerio de Ciencia, Tecnologa y Medio Ambiente (CITMA, Cuba)
(20092011) to J.L.C.; Grant 176351 from CONACYT to J.O.; and
Red del Programa Iberoamericano de Ciencia y Tecnologa para el
Desarrollo (CYTED-PROMAL, 210RT0398) Protemica y Quimiogenmica de Inhibidores de Proteasas de Origen Natural con
Potencial Teraputico en Malaria to MC. The authors thank
Dr. Isel Pascual, Centro de Estudio de Protenas, Facultad de Biologa, Universidad de La Habana, Cuba, for helpful advice during
the conception of this work and critical reading of the manuscript;
Dr. Emir Salas, Universidad Nacional de General San Martn,
Argentina, for useful suggestions for the design of rpfam1 gene
and expression experiments; Fidelia Romero, Instituto de Biotecnologa, UNAM, for assistance in the Molecular Biology techniques; and Lic. Annamil lvarez, Fundacin Instituto de Estudios
Avanzados, Venezuela, for technical assistance during the Western
blot assay.
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Please cite this article in press as: J. Gonzlez-Bacerio et al., High-level expression in Escherichia coli, purication and kinetic characterization of Plasmodium
falciparum M1-aminopeptidase, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.08.002