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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2007.00347.x |
Expression and Characterization
of Recombinant Thermostable Alkaline Phosphatase from a Novel Thermophilic
Bacterium Thermus thermophilus XM
Jianbo LI1, Limei
XU2,
and Feng YANG2*
1 School of Life Sciences, Xiamen
University, Xiamen 361005, China;
2
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography,
State Oceanic Administration, Xiamen 361005, China
Received: April 10,
2007
Accepted: June 20,
2007
This work was supported
by a grant from the China Ocean Mineral Resources R & D Association
*Corresponding
author: Tel, 86-592-2195276; Fax, 86-592-2085376; E-mail,
[email protected]
Abstract A gene (tap) encoding a thermostable
alkaline phosphatase from the thermophilic bacterium Thermus thermophilus
XM was cloned and sequenced. It is 1506 bp long and encodes a protein
of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison
of the deduced amino acid sequence with other alkaline phosphatases showed that
the regions in the vicinity of the phosphorylation site and metal binding sites
are highly conserved. The recombinant thermostable alkaline phosphatase
was expressed
as a His6-tagged
fusion protein in Escherichia coli and its enzymatic properties were
characterized after purification. The pH and temperature optima for the
recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ºC.
As expected, the enzyme displayed high thermostability, retaining more than 50%
activity after incubating for 6 h at 80 ºC. Its catalytic function was
accelerated in the presence of 0.1 mM Co2+, Fe2+, Mg2+, or Mn2+ but was
strongly inhibited by 2.0 mM Fe2+. Under optimal conditions, the Michaelis
constant (Km) for cleavage of p-nitrophenyl-phosphate
was 0.034 mM. Although it has much in common with other alkaline phosphatases,
the recombinant thermostable alkaline phosphatase possesses some unique
features, such as high optimal pH and good thermostability.
Keywords gene expression; Thermus thermophilus XM; thermostable
alkaline phosphatase
Alkaline phosphatases (APases; EC 3.1.3.1) exist widely in nature from
microorganisms to mammals, and play an important role in fundamental
biochemical processes, especially phosphate transportation and metabolism [1–3]. They can be
applied extensively in diagnostics, immunology, and molecular biology as
sensitive biological markers in processes such as enzyme-linked immunosorbent
assay, Western blotting analysis, nucleic acid hybridization, and in situ
hybridization [4–6]. APase from Escherichia coli has been extensively studied
in terms of biosynthesis, structure, and catalytic mechanism [7–13]. E. coli
APase is located in the periplasmic space as a homodimer and each monomer
comprises 449 amino acid residues and contains two Zn2+ and one
Mg2+.
At present, E. coli APase and calf intestine APase are the
most commonly used, but their inherently low thermal resistance and shelf-lives
have restricted their further applications under some special circumstances,
for example, high temperature and high pH. Compared with common APase,
thermostable APase (TAPase) has many beneficial qualities, such as high
thermostability, high reaction rates, and excellent resistance to denaturation
or microbial contamination [14]. Due to these advantages, there has been
increasing attention to TAPase from thermophilic bacteria. So far, a number of
TAPases have been isolated and their corresponding genes have been cloned and
characterized from various sources, such as Thermus species, Thermotoga
neapolitana, Meiothermus ruber, Bacillus stearothermophilusi,
and Pyrococcus abyssi [15–25].
In this study, we cloned and sequenced a tap gene from T.
thermophilus XM, discussed the characteristics of the deduced primary
structure of the enzyme, expressed it in E. coli and provided a
preliminary characterization of the recombinant TAPase (rTAPase).
Materials and Methods
Identification of strain
A thermophilic bacterium, whose optimal growth temperature is in the
range of 70–75 ºC in Luria Bertani (LB) medium, was isolated from a hot spring
at Dongfu beach (Xiamen, China). After incubation in LB medium for 24 h at 75 ºC
with shaking at 150 rpm, the bacterial cells were harvested and the genomic DNA
was purified using the Wizard genomic DNA purification kit (Promega, Madison,
USA). To identify the isolate, the 16S ribosomal RNA (rRNA) gene was amplified
by polymerase chain reaction (PCR) using universal primers, 27F (5‘-AGAGTTTGATCCTGGCTCAG-3‘)
and 1492R (5‘-GGTTACCTTGTTACGACTT-3‘). The PCR product was
cloned into pMD-18T vector (TaKaRa, Dalian, China) and sequenced. The
nucleotide sequence homology was analyzed using the blast program (http://www.ncbi.nlm.nih.gov/blast).
Cloning of the tap gene
Based on the complete coding region of the TAPase genes from the
genus Thermus, the following specific primers were synthesized: tapN (5‘-GGGGGATCCAAGCGAAGGGACATCCTG-3‘,
sense primer, BamHI restriction site underlined); and tapC (5‘-GGGAAGCTTTTAGGCCCAGACGTCCTC-3‘,
antisense primer, HindIII restriction site underlined). The amplified
fragment of 1506 bp was obtained by PCR with genomic DNA of T. thermophilus
XM as the template, cloned into pQE30 vector (Qiagen, Hilden, Germany), and
further confirmed by sequencing. The 25 ml PCR mixture consisted of
100 ng genomic DNA of T. thermophilus XM, 0.4 mM each primer, 200 mM each dNTP, 2.5
mM Mg2+, 1.25 U LA-Taq DNA polymerase (TaKaRa), and the buffer
supplied by the manufacturer. The reaction was carried out for 25 cycles as
follows: denaturation at 94 ºC for 30 s; annealing at 58 ºC for 30 s; and
extension at 72 ºC for 2 min.
Sequence homology searches were carried out using the blast program. Sequence analysis and
alignment were carried out with dnaman
software (version 5.1; Lynnon BioSoft, Vaudreuil, Canada).
Expression and purification of
rTAPase
After incubation at 37 ºC overnight, E. coli XL-Blue
containing the recombinant plasmid pQE30-tap and the control (vector only) were
inoculated into new media at a ratio of 1:100. When the A600 was 0.6, the cultures were induced with 0.5 mM isopropyl-b–D-thiogalactoside
for an additional 6 h at 37 ºC. Cells were harvested by centrifugation, then
suspended in 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. After
sonication, the insoluble debris was removed by centrifugation at 18,000 g
for 20 min and the supernatant was heated for 20 min at 70 ºC. The resulting
precipitate was again removed by centrifugation. Subsequently, the His6-tagged rTAPase in the supernatant fraction was purified using
Ni-NTA metal-affinity chromatography according to the instructions of the
QIAexpressionist system (Qiagen, Valencia, USA). The purified rTAPase was
dialyzed against the buffer containing 10 mM Tris-HCl, pH 8.0, 50% glycerol,
and the protein concentration was determined using the CB protein assay kit
(Calbiochem, La Jolla, USA).
Gel electrophoresis and
Western blot analysis
Protein samples were separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis [26] then transferred onto a polyvinylidene
fluoride membrane (GE Healthcare, Princeton, USA) by semi-dry blotting at a
constant current of 0.5 mA/cm2 for 1.5 h. The membrane was
immersed in blocking buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 3% bovine
serum albumin, and 0.05% Tween-20) at room temperature for 1 h, followed by
incubation with mouse anti-His antibody (GE Healthcare; diluted 1:3000) in
blocking buffer at 4 ºC overnight. Subsequently, a secondary antibody, alkaline
phosphatase-conjugated goat anti-mouse immunoglobulin G (Promega) was added at
a dilution of 1:7500 in blocking buffer at room temperature for 1 h, then the
immunoblot signals were detected using NBT-BCIP substrate (Roche, Mannheim,
Germany) in detection solution (50 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl2, pH 9.5).
Measurement of rTAPase
activity
The enzymatic reaction was carried out at 75 ºC for 10 min in 1.0 ml
appropriate buffer containing 6 mM p-nitrophenyl-phosphate disodium salt
hexahydrate (pNPP; Amresco, Solon, USA) and 1.5 mg of rTAPase, and terminated
on ice. The activity of rTAPase was measured spectrophotometrically at 410 nm.
To correct the rate of non-enzymatic hydrolysis of pNPP, the reaction
mixture without enzyme was used as a reagent blank. One unit of activity was
defined as the amount of enzyme required to release 1 mmol of p-nitrophenol
from pNPP in 1 min under the above assay conditions.
Characterization of rTAPase
The rTAPase was characterized by optimum pH, optimum temperature, thermostability,
and the effect of metal ions on its activity. Unless otherwise indicated, the
following experiments were carried out using the reaction conditions described
in the above section and the data were presented as the mean values of at least
three independent experiments.
To assess the effect of pH, rTAPase activity was measured at
various pH values ranging from 7.5 to 13.0. Tris-HCl buffer (100 mM) was used
in the pH range 7.5–9.0, and 100 mM diethanolamine buffer was used in the range 8.5–13.0.
The optimum temperature was measured by carrying out the rTAPase
activity assay at temperatures ranging from 40 ºC to 95 ºC at intervals of 5 ºC
and at pH 12.0. To determine the thermostability, the enzyme was incubated for
6 h at different temperatures (50 ºC, 65 ºC, 80 ºC, and 95 ºC). Samples were
taken out every 30 min and the residual activity was then measured as described
above.
To test the effect of metal ions, rTAPase activity was determined by
measuring the residual activity after incubation of the enzyme solutions with
various metal ions such as CaCl2, CoCl2, CuSO4, FeSO4, MgCl2, MnSO4, and ZnSO4 at 0.1 mM or 2.0 mM concentration.
Michaelis constant (Km) of
rTAPase
The Km value of rTAPase was determined
under optimum conditions using different concentrations of pNPP from 0
to 6 mM. A typical plot was obtained when v was plotted against v/[S]
(v, initial rate; [S], substrate concentration), according to the
method of Eadie-Hofstee and the Km value
was estimated by linear regression from it.
Results
Cloning and sequencing
analysis of the tap gene
At present, analysis based on the 16S rRNA gene sequence is
considered to be a standard in bacterial identification. For the thermophilic bacterium
from Xiamen, its 16S rRNA gene was amplified and sequenced (GenBank accession
No. DQ647385). Blast analysis
revealed that the strain had 99% similarity with T. thermophilus HB8
(GenBank accession No. AP008226). Based on sequence homology, the strain was
considered to be a T. thermophilus sp., so it was named T.
thermophilus XM.
A DNA fragment (approximately 1.5 kb) was amplified by PCR using tap
gene primers and sequenced. Sequence analysis revealed that the gene consisted
of 1506 bp (GenBank accession No. DQ645419) and was predicted to code a
polypeptide of 501 amino acids with a calculated molecular mass of 54.7 kDa and
isoelectric point of 8.7. The result from the DNA blast search showed that the tap gene shared over 93%
sequence identity with other tap genes from the genus Thermus. In
particular, it had the highest identity with T. thermophilus HB8 (99%).
Comparison of amino acid sequences of TAPase and other bacterial
APase
When the deduced amino acid sequence of the gene was compared with other
APases from thermophilic, mesophilic, and psychrophilic bacteria, TAPase had
the highest identity with that from genus Thermus, such as T.
thermophilus HB27 (99.6%) and T. caldophilus (98.4%), followed by M.
ruber APase (64.3%). Identity was lower with Thermotoga neapolitana APase
(27.3%), Bacillus subtilis APase (24.2%), Antarctic psychrophilic
bacterium TAB5 APase (20.6%), and E. coli APase (18.2%). Although the
whole sequence homology is not high between different APases, the alignment
analysis showed that their active site regions involved in metal ion
coordination, phosphorylation, and phosphate binding are highly conserved (Fig.
1), except that the residues D153 and K328 (E. coli numbering) are
replaced by histidine (compared with E. coli APase).
Expression and purification of
rTAPase
The tap gene was cloned into a His6-tagged
expression vector pQE30, then the recombinant plasmid pQE30-tap and empty
vector pQE30 were transformed into XL-Blue. After induction with isopropyl-b–D-thiogalactoside,
total cellular proteins were separated on 10% sodium dodecyl
sulfate-polyacrylamide gel and visualized by staining with Coomassie Brilliant
Blue. A protein an with apparent molecular mass of approximately 55 kDa,
consistent with the predicted 54.7 kDa molecular mass of TAPase, was observed
in XL-Blue cells transformed with pQE30-tap [Fig. 2(A), lane 1] but
absent from empty vector pQE30 [Fig. 2(A), lane 2]. The His6-tagged protein was purified by Ni-agarose chromatography and its
concentration was determined to be 0.15 mg/ml. The purified product contained
two bands of molecular masses 55 and 43 kDa, the slower migrating band being
more abundant than the faster migrating band [Fig. 2(A), lane 3]. Western
blot analysis revealed that the 55 kDa protein was recognized specifically by
anti-His antibody, indicating that it was indeed rTAPase [Fig. 2(B)].
Properties of rTAPase
Generally, the pH value of reaction solution can significantly
affect enzyme activity. Like other APases, the rTAPase was activated under
alkaline conditions. Fig. 3 shows the pH profile of purified rTAPase
activity. The optimal pH range of the enzyme was found to be 11.5–12.5, and
maximal activity occurred at pH 12.0. It was nearly inactive below pH 7.5.
The purified rTAPase had its maximal enzymatic activity at 75–80 ºC, and
approximately 50% of the activity was retained at temperatures above 40 ºC and
below 85 ºC, indicating that the enzyme is capable of functioning over a broad
temperature range (Fig. 4). In order to better estimate the effect of
temperature on enzyme activity, thermostability was determined by incubating
the rTAPase at temperatures ranging from 50 ºC to 95 ºC for different time
points. As shown in Fig. 5, the rTAPase can be characterized as a
moderately thermostable enzyme: over 50% of the activity was retained even
after 6 h at 80 ºC. However, the activity was sharply decreased after treatment
for 1.5 h at 95 ºC.
As APases are metalloenzymes [2], metal ions as co-factors might
play important roles in enzymatic reactions. The effects of some divalent metal
ions on the rTAPase activity are shown in Table 1. Mg2+ is the most optimal for enzymatic reaction activity, but Zn2+, Ca2+, and Cu2+ show an
inhibitory effect. Co2+, Fe2+, and Mn2+ (0.1 mM) can also significantly enhance the activity of rTAPase,
especially Co2+, however, there is a strong inhibition in the
presence of 2 mM Fe2+. As expected, the enzymatic activity of
rTAPase was strongly influenced in the presence of the chelating agent EDTA
(data not shown).
The Km value of the rTAPase for pNPP
was estimated to be 0.0340.009 mM (Fig. 6), similar to those previously
reported in other bacteria, such as T. caldophilus APase (0.036 mM)
[20], M. ruber APase (0.055 mM) [23], and E. coli
APase (ranging from 0.021 mM to 0.040 mM) [28,29], suggesting that the initial
velocity of the reaction catalyzed by APases from both thermophilic and
mesophilic bacteria is close.
Discussion
APases from thermophilic microorganisms are becoming more and more
interesting in diagnostics, immunology and molecular biology such as
non-radioactive labeling, due to their inherent advantages which are more
resistant to thermal denaturation during preparation or labelling procedures,
easier to transport and store, and longer half-life of enzyme activity.
In this study, a
tap gene was cloned from
T. thermophilus XM. Although the sequence homology is very low between
different APases, the amino acid residues in the active site were highly
conserved (Fig. 1). All ligands to Zn1, Zn2 and Mg, S102 and R166 are
absolutely conserved in all APases. In E. coli APase, Zn1 is coordinated
by two imidazole bases of H331 and H412, as well as one carboxyl of D327. Zn2
is bound by two carboxyls of D51 and D369 along with one imidazole base of
H370. Mg is bound by residues D51, T155, and E322 [12]. However, the secondary
ligands of the Mg ion, that is, D153 and K328, are replaced by histidine in T.
thermophilus XM APase (Fig. 1). D153 is a part of the Mg ion binding
site, and K328 is important for phosphate binding in E. coli APase. The
same substitutions were found in T. thermophilus HB27, T. caldophilus,
M. ruber, and mammalian APases. Previous studies showed that the
replacement of D153 resulted in an enhancement of enzymatic activity when the
Zn2+ bound in this site is replaced by Mg2+ [27].
The characterization studies showed that rTAPase is one of the most
characterized alkaline APases so far identified. By analogy with the mammalian
APase, which have higher pH optima than the E. coli APase, we can
speculate that the substitution of amino acid residues corresponding to D153
and K328 in rTAPase by histidine is responsible for the more alkaline activity
profile of the rTAPase [30]. However, the reason for alkaline phosphatase’s
resistance to extreme pH remains unclear. In fact, we are trying to reveal the
inherent mechanism of this resistance to extreme alkaline in another
experiment.
APase is a metalloenzyme, and different metal ions could have
different influences on rTAPase activity. Many studies showed that the same
metal ion has different effects (enhancer or inhibitor) on the activity of
APases from different sources, for example, Zn2+ could
be an inhibitor for M. ruber APase but be an enhancer for Thermotoga
neapolitana APase; Co2+ could enhance the activity of T.
thermophilus XM APase but inhibit the activity of M. ruber APase [22–25]. We
hypothesize that the phenomenon might be the result of the differences between
the active sites conformation of different APases. As shown in Fig. 1,
although almost all metal coordinating amino acid residues in the active sites
are conserved, the residues close to metal binding sites are variable from
different sources. It suggested that these varieties might result in some
conformational changes to affect the binding affinity for different metal ions.
In
summary, a tap gene from T. thermophilus XM was successfully
cloned and expressed in E. coli. The recombinant enzyme shared common
characteristics of alkaline phosphatase and displayed good thermostability. In
addition, it also revealed a unique property in that it showing a strong
competence for high alkaline conditions compared with other APases. These
characteristics suggest that the enzyme has potential value in experimental
science. For instance, it could be applied in nucleic acid hybridization and in
situ hybridization under high temperature and alkaline environments instead
of APases from other sources.
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