Supplementary Material
Chemicals and reagents
L-asparagine (L-Asn), flavine adenine dinucleotide (FAD), nicotinamide adenine
dinucleotide (NADH), horseradish peroxidase (HRP type X, 386 U/mg, Mr ~ 44 kDa),
glutamate dehydrogenase from bovine liver (GDH, 44 U/mg, Mr ~ 56 kDa), αketoglutarate, o-dianisidine, L-aspartate (L-Asp), and human serum (from male AB
plasma) were obtained from Sigma Aldrich (St. Louis MO, USA). Amplex Red (AR) was
purchased from Cayman chemicals (Michigan, USA). Yeast extract, peptone, agar,
ampicillin, and Isopropyl-beta-D-thiogalactoside (IPTG) were purchased from Applichem
(Gatersleben, Germany). KAPA HiFi polymerase was obtained from PeqLab (Erlangen,
Germany), restriction enzymes and T4 DNA ligase from NEB (Ipswich, MA, USA). Gel
extraction and PCR purification kits, and nickel agarose for affinity purification of polyhistidine-tagged proteins were from Macherey Nagel (Düren, Germany), plasmid
purification kit from Fermentas (Thermo Fisher Scientific, Germany), genomic DNA
preparation kit from Qiagen (Hilden, Germany), gel filtration chromatography column
Superdex 200 from Pharmacia/GE Health Care Life Sciences (Uppsala Sweden).
Coomassie Brilliant Blue G-250 (Bradford reagent) for protein assays was from Roth
(Karlsruhe, Germany).
Cloning, expression and purification of L-aspartate oxidase and periplasmic Lasparaginase from E.coli.
Since L-aspartate oxidase (L-AspOx) is commercially not available, we cloned the E.coli
gene (nadB) and produced milligram amounts of the enzyme recombinantly. The open
reading frame (ORF) of L-AspOx consisting of 1623 bp, was amplified by PCR using as
template genomic DNA isolated from the E.coli XL1-Blue strain. Unique NdeI and
BamHI restrictions sites (underlined below) were incorporated at the 5’- and 3’- ends of
the respective oligonucleotides (5’-GGAATTCCATATGAATACTCTCCCTGAACATTC3’, and 5’-CGCGGATCCTTATCTGTTTATGTAATGATTGC-3’). The PCR mixture (50
μL) consisted of 1 μg genomic DNA, oligonucleotide mix (10 pmol each), KAPA high
fidelity buffer, dNTPs (0.2 mM each), and 1 Unit KAPA HiFi DNA polymerase. The
reaction was performed at initial temperature of 95 °C for 3 min, followed by 25 cycles of
the next steps: at 98 °C for 20 s, 60 °C for 60 s, and 72 °C for 30 s. The reaction was
terminated after a 3’-end polishing step of 5 min at 72 °C. The PCR product was
agarose gel-purified, digested with NdeI and BamHI HF, and ligated overnight at 16 °C
into a pET14b-SUMO vector [1] using T4 DNA ligase. The ligation mixture was used to
transform DH5α E.coli cells, and positive clones were first identified by colony PCR
screening. Extracted plasmid DNA was digested with NdeI and BamHI HF to verify
correct size of the cloned fragment, and finally the gene insert was sequenced (SeqLab,
Goettingen, Germany).
The expressed protein construct includes an N-terminal six-histidine tag, followed by a
yeast SUMO (Small Ubiquitin-related MOdifier) tag which has proven to ameliorate
heterologous protein solubility and stability [2]. E.coli BL21(DE3) C41 cells (Lucigen,
Madison, USA) were cultured overnight at 37 °C in LB medium supplemented with 200
μg/ml ampicillin. A fraction of this culture was used to inoculate fresh 2xYT culture
medium (dilution 1:100) supplemented with 200 μg/ml ampicillin. When O.D. 600 reached
~ 0.5-0.7, expression was induced by adding IPTG to a final concentration of 1 mM.
After incubation at 37 °C for 8 hours, the cells were harvested by centrifugation (20 min
at 5,000g), resuspended in nickel agarose binding buffer A (50 mM Na 2HPO4, 0.5 M
NaCl, 10 mM imidazole, pH 8), and ultimately lysed by ultrasonication. The cell lysate
was centrifuged (45 min at 17,200g), the resulting supernatant mixed with preequilibrated nickel agarose beads, and incubated at 4 °C for 3 hours in a rotating
beaker. The slurry mixture was filled in 5 mL polypropylene columns, dried by gravity,
and the agarose beads were then washed with 25 bed volumes of buffer B (50 mM
Na2HPO4, 1 M NaCl, 25 mM imidazole, pH 8). Bound protein was eluted from the
column by applying 300 mM imidazole in buffer B. All purification steps were performed
at 4 °C in a cold room. Eluted fractions were mixed, and buffer was exchanged against
50 mM Na2HPO4, 100 mM NaCl, pH 7.5, using a PD-10 column (GE Healthcare). Eluted
protein was incubated with yeast SUMO protease (molar ratio 1:100) at 30 °C for 2
hours to cleave the N-terminal tag. This treatment resulted in the final intact L-AspOx
with predicted Mr ~ 60.3 kDa (apparent Mr on SDS-PAGE ~ 60 kDa). The protein
solution was concentrated using ultrafiltration tubes, 10,000 MWCO (Sartorius,
Goettingen, Germany). In the final purification step, the enzyme was passed through a
Superdex 200 column to remove the cleaved tag, SUMO protease, and impurities
remaining after the affinity purification step. Protein purity, as evaluated by SDS-PAGE,
was more than 95%. The protein was aliquoted, mixed with glycerol (25% final
concentration), and stored at -20 °C until further use. The overall protein yield for LAspOx was estimated to be approximately 40 mg per L of 2xYT growth medium with a
specific activity ~ 0.05 U/mg as determined by applying the o-dianisidine assay
described elsewhere [3], which was also used for the kinetic characterization of this
enzyme (Fig. S3).
Periplasmic mature E.coli L-ASNase (ansB) was cloned using the primer pair 5’GGAATTCCATATGTTACCCAATATCACCATTTTAGC-3’
and
5’-
CGCGGATCCTTAGTACTGATTGAAGATCTGCTGG-3’, expressed, and purified in a
similar way. The protein amount obtained out of 1 L 2xYT medium was ~ 15 mg with a
specific activity of 1 U/mg as determined by the NADH-dependent assay. The predicted
Mr of the mature E.coli L-ASNase is 34.5 kDa, while on SDS-PAGE it appears to run
somewhat faster (~ 32 kDa).
Figures
Figure S1. SDS-PAGE (15%) analysis of purified E.coli L-AspOx (calculated Mr: 60.3
kDa).
Figure S2. SDS-PAGE (15%) analysis of purified mature E.coli L-ASNase (calculated
Mr: 34.5 kDa).
Figure S3. Steady-state turnover rates (kobs) of L-AspOx determined by applying the
chromogenic o-dianisidine-based assay [3]. Reactions were performed at 25 °C in 1 ml
PBS, containing 20 μM FAD, 100 nM HRP, 10 μg/ml o-dianisidine, 65 nM (~ 5 μg) LAspOx, and variable amounts of substrate. Kinetic constants (see text) were calculated
by
non-linear
regression
of
the
Michaelis-Menten
(Igor
Pro,
Wavemetrics).
Measurements were performed in triplicate (n = 3), and values are expressed as means
± SD.
Figure S4. Fluorescence intensity as a function of AR concentration. The L-Asn
concentration was kept constant at 1 μM, while L-ASNase concentration was ~ 4.6 nM.
The fluorescence signal was recorded after 30 min in each case, (n=3, ± SD)
Figure S5. Time course of the L-ASNase reaction monitored at discrete time points
using the fluorescence assay. Concentrations: 10 μM L-Asn, 50 μM AR, ~ 4.6 nM LASNase, (n=3, ± SD). Error bars are not shown when they are smaller than the
symbols.
Figure S6. Real-time monitoring of the three-step enzymatic reaction to detect Lasparaginase activity using a plate-reader (Molecular Devices, SpectraMax Paradigm;
wavelength settings were 532 nm for Ex, and 592 nm for Em). The four reactions took
place simultaneously in a 96-well plate in a final volume of 100 μL at the following
concentrations: 10 μM L-Asn, 50 μM AR; L-ASNase concentrations were as follows: (●):
0 nM, (■): 1 nM, (▲): 2 nM, and (◇): 4 nM.
Figure S7. Enzyme-concentration response using human serum supplemented with LASNase. 200 μL of human serum were spiked with 50 μg L-ASNase previously diluted
in PBS, and mixed thoroughly. Subsequently, aliquots were used to detect enzymatic
activity in a final volume of 100 μL using standardized conditions as described in the
main text for the fluorescence mode, except from the following modifications of
concentrations and settings: 100 μM L-Asn and 500 μM AR, Ex. bandwidth at 1 nm,
while Em. bandwidth was kept at 2.5 nm. Fluorescence signals were corrected by
subtracting the control values (no L-Asn) from all samples, (n=3, ± SD).
Figure S8. Steady-state kinetics of L-ASNase applying the NADH-dependent assay.
Reactions were performed at 25 °C in 1 ml PBS, containing 0.25 mM NADH, 0.25 mM
α-ketoglutarate, 35 nM GDH and 7 nM (~ 250 ng/ml) L-ASNase. Data points are
represented as means of triplicate measurements, and error bars are standard
deviations of three separate reactions. Kinetic constants were calculated by non-linear
regression of the Michaelis-Menten equation (Igor Pro, Wavemetrics) and are shown in
Table S1.
Table S1. Steady state-kinetic parameters (n=3, ± S.D.) for E.coli L-ASNase
determined by applying the AR or the NADH-dependent assay.
Kinetic parameter
Amplex Red assay
NADH assay
Km
22 ± 1.2 (μM)
21 ± 1.5 (μM)
kcat
12.1 ± 0.5 s-1
11.5 ± 0.4 s-1
kcat/Km
5.5 x 105 M-1 s-1
5.4 x 105 M-1 s-1
Z’ factor determination
In order to evaluate and validate the newly developed assay for L-ASNase, we
determined the Z’ factor [4] which is defined as follows:
where μc+ and μc- are the means of the positive and negative control values,
respectively, and σc+ and σc- are the standard deviations of the corresponding signals.
The statistical parameters were calculated from six samples containing 10 μM Lasparagine for the positive control, and six ones which contained all components except
from L-Asn for the negative control. The final reaction solution contained 3.5 μΜ L-
AspOx, 10 μΜ FAD, 100 nM HRP, 50 μΜ AR and 4.6 nM L-ASNase (~ 16 ng/0.1 ml).
The Z’ factor was determined to be 0.77 suggesting that this is a very sensitive and
reproducible assay.
Some Notes and Tips
1) L-AspOx, a monomeric enzyme, has been highly overexpressed recombinantly in
E.coli. Instead of IPTG, one could use equally efficiently the cheaper “auto-induction”
medium which contains lactose as inducer. We found similar expression yields (>40
mg/L 2XYT medium) in both cases, using the pET-14b-SUMO vector. Such yields
could be easily achieved not only when BL21(DE3) C41 was used as expression
host, but also with other conventional E.coli strains such as BL21(DE3), BL21(DE3)
pLysS, or BL21(DE3) Rosetta.
2) AR is a light-sensitive dye, and therefore it is essential to avoid its exposure to light.
We observed that the reaction mixture (without L-ASNase to trigger the reaction)
remains stable, exhibiting a very low background fluorescence signal, for 3 hours at
room temperature in dark Eppendorf tube.
3) FAD is a crucial co-factor for the activity of L-AspOx; it is non-covalently bound to the
enzyme with 1:1 stoichiometry. However, it is not tightly bound (K ~ 1 μM), and
therefore a 3-fold molar excess of FAD provides a more stable environment for the
activity of L-AspOx.
4) Fresh preparation of all buffers is advantageous, as this can diminish undesired
background activity. A noteworthy example is the L-Asn stock solution. L-Asn, when
stored for long periods even at 4 °C, can spontaneously hydrolyze to L-Asp and
ammonia. This would cause a considerably higher initial background activity, since
the presence of L-Asp would initiate reaction steps two and three of the coupledenzyme system. Furthermore, in a series of experiments, we observed that AR
remained stable for 4 months when dissolved in DMSO at a final concentration of 50
mM.
5) For the purpose of detection of L-ASNase activity in serum, it is preferable to use
saturated L-Asn concentrations (for E.coli L-ASNase > 80 μM) and consequently
higher AR concentrations (five fold higher than L-Asn). This will decrease the
background reaction and improve the signal-to-noise ratio.
References
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L-Asparaginase: Insights into the Mechanism of Autoproteolysis and Substrate
Hydrolysis, Biochemistry 51 (2012) 6816−6826.
[2] T. Panavas, C. Sanders, T. R. Butt, SUMO Fusion Technology for Enhanced Protein
Production in Prokaryotic and Eukaryotic Expression Systems, Methods in Molecular
Biology, SUMO Protocols 497 (2009) 303-317.
[3] G. Tedeschi, S. Ronchi, T. Simonic, C. Treu, A. Mattevi, A. Negri, Probing the active
site of L-Aspartate oxidase by site-directed mutagenesis: Role of basic residues in
fumarate reduction, Biochemistry 40 (2001) 4738-4744.
[4] J.H. Zhang, T.D.Y. Chung, K.R. Oldenburg, A simple statistical parameter for use in
evaluation and validation of high throughput screening assays, J. Biomol. Screen. 67
(1999) 67-73.