From the Structural Biology Center, Korea Institute
of Science and Technology, Seoul, 136-791 Korea, the
§ Department of Agricultural Chemistry, Korea University
Seoul, 136-701 Korea, and the ¶ Department of Chemistry,
University of California, Berkeley, California 94720
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ABSTRACT |
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A gene encoding a serine-type protease has been
cloned from Aquifex pyrophilus using a sequence tag
containing the consensus sequence of proteases as a probe. Sequence
analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and
Ser-502) that form the catalytic site of serine-type proteases. The
size of the mature form (43 kDa) and its localization in the cell wall
fraction indicate that both the NH2- and COOH-terminal
sequences of the protein are processed during maturation. When the
cloned gene is expressed in Escherichia coli, it is weakly
expressed as active and processed forms. The pH optimum of this
protease is very broad, and its activity is completely inactivated by
phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h
at 105 °C, suggesting that it is one of the most heat-stable
proteases. The cysteine residues in the mature form may form disulfide
bonds that are responsible for the strong stability of this protease,
because the thermostability of the protein is significantly reduced in
the presence of reducing reagent.
Hyperthermophiles are organisms that grow optimally at 80 °C or
higher temperatures. Ever since they were identified at various geothermal locations, there has been interest in their evolutionary significance as well as their ability to thrive at temperatures in
which normal proteins would be denatured. From the ability to grow
autotrophically at high temperature and their position in the deepest
branch of the phylogenetic tree, hyperthermophiles are presumed to be
the closest living descendants of a universal ancestor of life (1, 2).
Except for two genera of eubacteria, Aquifex and
Thermotoga, all identified hyperthermophiles belong to
Archaea. Most of them are grouped as autotrophs that use inorganic materials such as carbon and nitrogen sources or heterotrophs that
exploit organic materials as nutrients (3). To degrade exogenous
organic compounds, heterotrophic hyperthermophiles have various
hydrolytic enzymes such as proteases and glycosidases. Autotrophic
organisms also contain proteases that may be involved in cellular
processes. The proteases from hyperthermophiles have been studied for
their biotechnological applications as well as for their metabolic
significance (4). Among the existing types of proteases such as
serine-, thiol-, or acid-type proteases, the majority of them belong to
a family of serine-type proteases (5-9). Some of them are processed
from a preprotein to a mature form and are localized at the cell wall
or are secreted. These proteases show heat stability or are resistant
to a high concentration of denaturing agents. The half-life measured at
around 100 °C varied from a few minutes for archaelysin (5) to
several hours for pyrolysin (6).
Aquifex pyrophilus is a microaerobic eubacterium that grows
optimally at 85 °C. This organism is also regarded as the most ancient bacterium because of its autotrophic nature and its deep location in the phylogenetic tree (10). Several genes homologous to
proteases have been identified from the genomic sequence of Aquifex aeolicus (11). In addition, sequence tags similar to protease-like genes such as the lon protease in
Escherichia coli allowed the recognition of the signal
peptidase and the serine-type protease from the sequence analysis of
the genomic DNA of A. pyrophilus (12). The role of the
serine-type protease in A. pyrophilus may not be involved in
the degradation of extracellular peptides because these compounds are
not utilized as carbon or nitrogen sources.
To characterize the serine-type protease of A. pyrophilus,
we have cloned the entire gene and determined its DNA sequence. The
cloned gene belongs to the subtilisin family and is localized in the
cell wall fraction of A. pyrophilus as a mature form of 43 kDa. When the gene is transformed into E. coli, it is
expressed as active and processed forms, although the expression level
is low. This protein shows extreme stability at high temperatures and
at a broad range of pH values.
Materials, Strains, and Cultivation--
Restriction
enzymes and T4 DNA ligase were obtained from Promega. Sequencing
primers were purchased from Bioneer (Seoul, Korea). A. pyrophilus was obtained from the German Collection of
Microorganism and Cell Culture (Deutsch Sammlung von
Mikroorganismen, Braunschweig, Germany) and was grown in a modified SME
medium (13) at 85 °C as described by Huber et al. (14).
The cells were inoculated into 20 ml of liquid medium in a 120-ml
bottle that was filled with 3 bars of hydrogen, carbon dioxide, and
oxygen at a ratio of 79.5:20:0.5 and grown for 12 h with moderate
shaking. Cells were harvested by centrifugation at 10,000 × g for 10 min, washed with 10 mM Tris-HCl, pH
8.0, and stored at
The E. coli strain used for plasmid DNA amplification was
DH5aH (supE44 DNA Isolation and Library Construction--
A.
pyrophilus genomic DNA was purified using a genomic DNA
purification kit (Qiagen, Hilden, Germany). For the construction of the
genomic Molecular Cloning and Sequence Analysis--
Standard procedures
were used for cloning and sequencing as described by Sambrook et
al. (15). Southern analysis was performed with an ECL kit from
Amersham Pharmacia Biotech (UK). The determination of nucleotide
sequence was carried out with an ABI 373 automatic DNA sequencer
(Applied Biosystems). The obtained DNA sequences were converted into
amino acid sequences in all six possible reading frames and were
compared with known proteins in the data base using a sequence
comparison program, BLAST (16). For multiple sequence alignment between
functionally related proteins, the SEQSEE (17) and MACAW programs (18)
were used. The putative membrane-spanning region was assigned using the
TMpred program (19).
Fractionation of A. pyrophilus--
Cell paste of A. pyrophilus was suspended in lysis buffer (50 mM
Tris-HCl, pH 8.0, 20 µg/ml lysozyme) and incubated for 10 min at
37 °C. The cells were sonicated for 2 min using a Branson Sonifier
450 (Branson Ultrasonic Co.). The crude extract was centrifuged for 20 min at 18,000 × g, and the pellet representing the
cell wall fraction was recovered. The proteins in the cell wall
fraction were extracted with 0.1% SDS at 95 °C for 20 min. The
membrane fraction was prepared from the crude extract using serial
centrifugation. The crude extract was centrifuged at 10,000 × g for 10 min, and the supernatant was further centrifuged at
100,000 × g for 1 h. The pellet representing the
membrane fraction was resuspended in 50 mM Tris-HCl, pH
8.0. For the preparation of peripheral or integral membrane proteins,
the membrane fraction was treated with 1.0 M NaCl or 0.5%
Triton X-100 and was centrifuged at 100,000 × g for
1 h.
Construction of Expression Vectors and Expression in E. coli--
Different regions of the open reading frame of the cloned
protease gene were amplified by the polymerase chain reaction with 5'
and 3' primers harboring NdeI and BamHI
restriction sites, respectively. The amplified DNA fragment and the
vector, pET21a (Novagen), were treated with NdeI and
BamHI, ligated, and transformed into E. coli
BL21(DE3) cells. The transformed cells were grown at 37 °C in LB
medium containing 100 µg/ml ampicillin with vigorous shaking to an
A600 nm of 0.7. Protein expression was induced
by adding 1 mM
isopropyl-1-thio- Antibody Production, Immunoprecipitation, and Western
Analysis--
For the preparation of an antibody specific to the
cloned gene product, a fragment of the open reading frame representing amino acids 102-494 was expressed in E. coli using the pET
system as described above. The expressed protein, recovered from
inclusion bodies, was separated by 10% SDS-polyacrylamide gel
electrophoresis (PAGE).1 The
protein band was excised from the gel, crushed, and injected into a
mouse for antibody production. Mouse serum was obtained 4 weeks after
injection. For Western analysis, proteins were separated on SDS-PAGE
and transferred to a polyvinylidene difluoride membrane (Bio-Rad) in
100 mM CAPS buffer, pH 10.0. The membrane was treated with
a 5% skim milk solution and then reacted with 1/2,000 diluted serum in
TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1%
Tween 20, pH 7.5) for 1 h at room temperature. After washing with
TBST buffer, protein bands that bound to the antibody were detected using a chemiluminescent reaction using the ECL Western blotting system
(Amersham Pharmacia Biotech, UK). Immunoprecipitation was carried out
according to the method described by Kessler (20). The immune serum was
added to 50 ml of the cell wall extract and incubated on ice for 1 h with occasional stirring. Then, 50 ml of Staphylococcus
aureus formalin fixed cells (Sigma) were added to the mixture and
incubated for 1 h at 0 °C. The mixture was centrifuged at 3,000 rpm for 10 min, and protease activity in the supernatant was further analyzed.
Purification of Protease Expressed in E. coli--
E.
coli cells that expressed the cloned gene were harvested at
4,000 × g for 10 min and resuspended in lysis buffer
without lysozyme (50 mM Tris-HCl, pH 8.0). The cells were
disrupted by sonication for 2 min, and the soluble fraction was
recovered by centrifugation at 17,000 × g for 20 min.
After removing heat-labile proteins in the crude extract by heat
treatment for 20 min at 95 °C, the soluble fraction was loaded onto
a 1.5 × 15-cm Q-Sepharose column (Amersham Pharmacia Biotech,
Sweden) equilibrated with 20 mM Tris-HCl, pH 8.0. A 200-ml
linear gradient from 0 to 1.0 M NaCl in equilibrating
buffer was used to elute the protease.
Assays--
Either a colorimetric method or a gel-staining
method was used to measure protease activity. For a colorimetric
method, samples were reacted with 0.5% azocasein as described by Cowan
et al. (5) with a slight modification. The reaction mixture
consisted of 100 µl of enzyme sample and 500 µl of 0.5% azocasein
dissolved in 50 mM potassium phosphate buffer, pH 9.0. After incubation at 85 °C for 30 min, the reaction was stopped by
the addition of 500 µl of 15% trichloroacetic acid, and the
absorbance value at 440 nm was measured using a UV-visible
spectrophotometer (Shimadzu, Kyoto, Japan). The gel staining assay was
carried out using 0.1% gelatin-containing gel as described by Kleiner
and Stetler-Stevenson (21). Proteins were assayed by the Bradford
method (22) using bovine serum albumin as a standard.
Cloning and Sequencing of a Serine-type Protease Gene of A. pyrophilus--
One of the Sequence Alignment of the Protease Gene of A. pyrophilus--
The
amino acid sequence of the cloned gene was compared with gene sequences
deposited in data banks using the BLAST program. The genes that show
strong sequence similarity to the cloned gene are serine-type
proteases. It has three canonical residues of aspartic acid (Asp-140),
histidine (His-184), and serine (Ser-502) that constitute the active
site of serine-type proteases. Amino acid sequences around these
residues are highly homologous to those of other proteases (Fig.
3A). The cloned gene has three regions of nonhomologous sequences at the NH2 and COOH
terminus and between His-184 and Ser-502 (Fig. 3B). The 150 amino acids at the NH2 terminus are assumed to be involved
in membrane translocation because the presence of a cluster of
hydrophobic amino acids and positive charges following them are well
matched to those of a bacterial secretion signal sequence. The
COOH-terminal nonhomologous region starts from amino acid 555. Interestingly, this region contains a putative transmembrane sequence
(596-615) at the COOH-terminal end (Fig. 3C). The sequence
of 150 amino acids between His-184 and Ser-502 does not show any
sequence similarity with any proteins in the data base. However, a
serine-type protease from Bacillus subtilis, Vpr (24), and a
cell envelope-localized serine protease from Lactobacillus
lactis, Wg2 (25), also have a nonhomologous sequence between the
active site residues, His and Ser. Although these sequences are not
homologous, the size of this nonhomologous sequence and the arrangement
in the gene are comparable.
Identification and Localization of the Cloned Gene Product in A. pyrophilus--
To confirm whether the cloned gene is expressed in
A. pyrophilus, Western analysis was performed on the crude
extract of A. pyrophilus with an antibody raised against a
truncated protein of the cloned gene. A 43-kDa protein from the crude
extract of A. pyrophilus was strongly detected by the
antibody along with two faint bands migrating at 55 and 64 kDa. These
bands are mainly observed in the cell wall fraction, and only small
portions are detected in the cytosolic fraction (Fig.
4A). These results indicate that the cloned gene is expressed in A. pyrophilus as a
precursor form, translocated to the cell wall, and processed to a
mature form of 43 kDa. The 64- and 55-kDa bands are probably
intermediate forms of the processing step. When the proteases in the
extract of A. pyrophilus were observed by the gel staining
assay, at least three positive proteases appeared in the cell wall
fraction. Among them, the 43-kDa protease showed the highest
proteolytic activity (Fig. 4B). Treatment of the gel with 1 mM phenylmethylsulfonyl fluoride abolished most of the
protease activity of the 43-kDa protein,2 indicating that the
43-kDa protease is a serine-type protease. To examine whether the
43-kDa protein observed in the Western analysis and in the gel-staining
activity assay is the same protein, immunoprecipitation analysis was
performed. The 43-kDa protease selectively cross-reacted with the
antibody specific to the cloned gene product and was precipitated by
S. aureus cells (Fig. 4C). These results show
that the 43-kDa protein encoded by the cloned gene is the major serine
protease in the cell wall fraction.
The cloned gene has a putative transmembrane sequence at the COOH
terminus. To examine whether the gene product is a membrane protein,
the membrane fraction was prepared from the cell extract of A. pyrophilus. Although the 43-kDa protein that cross-reacted with
the antibody was mainly observed in the cell wall fraction, a
significant amount of the protein was detected in the membrane fraction. To determine whether the protein is a peripheral or integral
membrane protein, extracted protein treated with 1.0 M NaCl
or 0.5% Triton X-100 was examined. Treatment with high salt did not
wash out the protein from the membrane, and only a small fraction of
protein was eluted from the membrane by detergent (Fig.
5). Failure of extraction by detergent
suggests that the 43-kDa protein is not a membrane protein. The protein
observed in the membrane fraction may be because of contamination
during cell fractionation.
Expression of the Protease Gene of A. pyrophilus in E. coli--
Attempts to purify the 43-kDa protease from A. pyrophilus were not successful because of low expression and
difficulty in cell growth. When the gene was expressed in the E. coli BL21(DE3) cell using a pET21a vector, the expression level
was very low, and the host cells stopped growing and lysed. When the
coding regions starting from the methionine at the 1st, 50th, or 102nd codon were expressed in E. coli, the proteins were expressed
at low levels and showed a toxic effect on the host cells.2
Expression of the coding region from the methionine at the 50th codon
showed the lowest toxic effect, and the expressed protein was
characterized. The expressed protein was not distinctively observed in
Coomassie-stained PAGE (Fig. 6A); however, it was observed
as multiple bands whose sizes were in the range of 34-55 kDa by
Western analysis (Fig. 6B).
The size of the expressed protein was reduced after heat treatment
(Fig. 6B). This result indicated that the cloned protease
expressed in E. coli undergoes self-processing.
Characteristics of the Protease--
To examine the properties of
the cloned protease, the protein expressed in E. coli was
partially purified by anion exchange chromatography and characterized.
In addition, the thermostability and pH optimum of the native protease
in the cell wall fraction of A. pyrophilus were also
investigated. The activity of the native protease was highest at
85 °C and pH 7-9 (Fig. 7,
A and B). When protease activity of the expressed
protein in E. coli was investigated using azocasein as a
substrate, it also showed a pattern similar to the native protease. The
maximum activity was measured at 85-95 °C, and the activity
gradually decreased as the temperature decreased (Fig.
8A). The optimum pH for the
protease activity was obtained at pH 9 (Fig. 8B). At the
same pH, the protease seemed slightly more active in phosphate buffer
than in Tris-HCl buffer. The stability of the expressed protein was
examined by measuring the decrease in activity after incubation at high
temperature. When the protein was incubated at 85 °C, it was slowly
inactivated, and its half-life was calculated to be 90 h (Fig.
9A). At 105 °C, the
activity of the protease decreased by 50% after incubation for 6 h (Fig. 9B). These results indicate that the expressed
protein is one of the highest thermostable proteases.
Sequence Characterization--
The cloned protease gene has
characteristics similar to those in the subtilisin family, the second
largest class of serine-type proteases (26). Although a canonical
ribosome-binding sequence that has been found in a manganese-superoxide
dismutase gene of A. pyrophilus (27) has not been found in
the cloned gene, the first methionine codon of the open reading frame
was proposed as the initiation site of translation because it was
located downstream of the putative promoter sequences. The size of the
native protein was smaller than the calculated size based on the
deduced amino acid sequence, indicating that the gene was translated as
a precursor protein and processed to a mature form. Many subtilisin
family proteases are translated as the prepro-form containing a
secretion signal sequence. The signal sequence (presequence) was
processed after translocation through the periplasmic membrane, and the following sequence (prosequence) was further processed to become the
mature form (28). In some cases, the prosequences were located at both
the NH2 and COOH termini of the protein and were cleaved during the maturation process (29). The 21 hydrophobic amino acids at
the NH2 terminus of the open reading frame have
characteristics found in signal sequences (30). Besides the signal
sequence, portions of the NH2- and COOH-terminal sequences
should be processed. The distance between the conserved sequences
containing aspartate (Asp-140) and serine (Ser-502) is about 385 residues, whose calculated size is slightly smaller than the size of
the mature form. Hence, the 110-130 residues at the NH2
terminus and the 80-100 residues at the COOH terminus of the precursor
form should be processed to generate a 43-kDa protein. It is
interesting that a putative membrane spanning sequence is present at
the presumed COOH terminus prosequence (Fig. 3C). A
transmembrane sequence at the COOH terminus of a protease that anchors
the protein to the membrane has been found in the Wg2 protease from
L. lactis (25). The hydrophobic sequence at the COOH
terminus may serve as a membrane anchor. In that case, the protein may
be attached to the membrane transiently because most of the gene
product was present as a processed form in the cell wall fraction.
Physiological Role of the Protease--
The function of the cloned
protease in the cell is unclear. The protease may not be used for
degradation of exogenous nutritional peptides, because A. pyrophilus is a strict autotroph. The physiological role of this
protein may be related to its cellular localization. A. pyrophilus contains a very stable proteinaceous layer, called the
S-layer, that is present outside of the outer membrane (14). The
S-layer of hyperthermophiles consists of heat-stable proteins, and it
is assumed to protect the cell from extreme environments such as high
temperature (31). Electron microscopic analysis has shown that a very
stable protease has been attached to the middle of the long stem that
forms the S-layer (7). We presume that the cloned protease is
associated with the S-layer proteins of A. pyrophilus,
because the protein is tightly associated to the cell wall where the
S-layer is present. However, whether the protease is involved in the
protein degradation process or serves as a building block of the
S-layer structure is currently unknown.
Factors Contributing to the Thermostability of the
Protease--
The cloned protease is extremely stable to heat. The
half-life of the expressed protein at 105 °C (6 h) suggests that
this enzyme is one of the most heat-stable proteases. Structural
analysis has shown that one of the major factors that stabilizes
proteins from hyperthermophiles is ionic bonds or salt bridges between charged amino acids (32). In addition, proteins from hyperthermophiles have more charged amino acids than proteins from mesophiles or thermophiles. A higher occurrence of charged amino acids is also observed in the proteins from A. pyrophilus (12). The
percentage of aspartate, glutamate, arginine, and lysine of the cloned
protease is about 20%. This is similar to the value found in pyrolysin from Pyrococcus furiosus and is much higher than proteases
from mesophiles or thermophiles (Table
I). These charged residues may be engaged
in the formation of salt bridges.
Another factor that contributes to the stability of the protease is
disulfide bonds. There are a total of nine cysteine residues in the
precursor form of the cloned protein and eight residues in the
predicted mature form. Most of these cysteine residues form disulfide
bonds that stabilize the protein because treatment with 1 mM dithiothreitol reduced the heat resistance of the
protein at 85 °C to less than 2 h.2 Hence,
disulfide bonds contribute to the extremely high stability of the
protein. Among 170 subtilisin family proteases, only a few, such as
aqualysin I, have cysteine residues that may form disulfide bonds (33).
Also, serine-type proteases from hyperthermophiles discovered so far do
not have cysteine residues except aerolysin and STABLE (7, 33).
Enhancement of thermal stability by introduction of intramolecular
disulfide bonds has been reported in phage T4 lysozyme (34) or
subtilisin (35); however, stabilization of the protein by disulfide
bonds is an unusual case among the serine-type proteases from
hyperthermophiles. The identity of the disulfide bonds that contribute
to the stability of the cloned protease is of current interest.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
80 °C until further usage.
lacU169(
80 lacZ
M15) hsdR17 recA1 endA1
gyrA96 thi-1 relA1). E. coli XL1-blue MRA P2
(
(mcrA)18
(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-l relA1
gyrA96 lac P2 lysogen) was used for
phage propagation, and
E. coli BL21(DE3) (E. coli B
F
dcm ompT hsdS(rB-
mB-) gal
(DE3)) was used for protein expression.
library, 20 µg of genomic DNA from A. pyrophilus was partially digested with HindIII and
ligated with 5 µg of
DASHII digested with HindIII. The
ligated DNA was packaged using a packaging kit (Stratagene) and
amplified in E. coli MRA (P2) in which only phages
containing foreign DNA were propagated. About 4,000 primary plaques
were obtained and further amplified. DNAs from
clones was isolated
using a Qiagen
DNA extraction kit.
-D-galactopyranoside for 3 h.
Cells were harvested by centrifugation at 4,000 × g
for 10 min.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
clones from the genomic DNA library of
A. pyrophilus hybridized to the sequence tag (Fig.
1A, AQpBB007) that contained a
sequence similar to that found in serine-type proteases (12). Southern
analysis indicates that the sequence tag used as a probe is located in
the middle of the cloned DNA. The sequence of the 2.1-kilobase region
including the sequence tag region has been determined in both strands
(Fig. 1B). In the sequenced region, there is only one open
reading frame (ORF) larger than 100 amino acids. This ORF contains the
serine-type protease consensus sequence present in the sequence tag.
The nucleotide sequence and deduced amino acid sequence of the ORF are
shown in Fig. 2. Upstream from the
beginning of the ORF, consensus bacterial promoter sequences were
found. The TATAAT and TTGAAG sequences at 24 and 52 bases upstream from
the open reading frame are homologous to the
10 (TATAAT) and
35
(TTGACA) promoter sequences (23). Downstream of the putative promoter
sites, there are four ATG codons (at the first, 50th, 102nd, and 121st
codon) that are candidates for translation initiation sites of the
gene. None of them has a distinct ribosome-binding sequence (AGGAGG) in
the adjacent 5'-region, except the purine-rich sequence (AACAAG)
located at the 8 bases upstream from the first ATG codon. In addition,
the amino acid sequences translated between the first and fourth ATG
codon are not conserved among proteases. However, the hydrophobic
stretch of 20 amino acids starting right after the first ATG codon has a characteristic bacterial secretion signal. Because the gene product
is mostly found in the cell wall fraction (see below), the first ATG
codon may serve as a translation initiation site and the hydrophobic
sequence may serve as a targeting signal. For these reasons, we have
tentatively assigned the first methionine as the start site for
translation. This ORF codes for a protein of 619 amino acids (69-kDa
protein).
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Fig. 1.
Probe sequence and restriction map of an
A. pyrophilus protease gene. A, the
nucleotide sequence of the sequence tag AQpBB007 around the active
site. The deduced amino acid sequence from the tag and the
corresponding region of aqualysin I (NCBI accession no. 94701) from
Thermus aquaticus YT-1 are compared. B,
restriction map of the 11-kilobase (kb) fragment containing
the A. pyrophilus protease gene. The open reading frame is
indicated by a thick line, and the sequence
regions are indicated by arrows under the restriction map.
The signal sequence and a putative membrane spanning sequence are
represented by black boxes, and the predicted
mature form is indicated by a gray box. The
numbers above the boxes indicate the positions of the amino
acid residues. E, EcoRI; H, HindIII;
S, SacI.
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Fig. 2.
Nucleotide and amino acid sequence of the
cloned protease gene of A. pyrophilus. The numbering is
started from the ATG codon at the +1 position. The putative
transcription sites are enclosed in a box. Amino acids in
black boxes represent the catalytic residues commonly found
in a serine-type protease. The putative signal sequence is
underlined. The probe sequence is indicated as a
dotted line.
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Fig. 3.
Sequence comparison of the cloned gene.
A, amino acid (a.a.) alignment of proteases
around three canonical residues (*). Identical and homologous residues
are indicated by black and gray boxes,
respectively. 1:Apy, A. pyrophilus;
2:Bsu, Vpr from B. subtilis (NCBI accession no.
135023); 3:Lla, Wg2 from L. lactis (NCBI
accession no. 472836); 4:Sma, STABLE from
Staphylothermus marinus (NCBI accession no. 137456);
5:Pfu, pyrolysin from P. furiosus (NCBI accession
no. 1556463); 6:Pae, aerolysin from Pyrobaculum
aerophilum (NCBI accession no. 2147085); 7:Taq,
aqualysin from T. aquaticus (NCBI accession no. 94701),
8:Tvu, thermiatase from Thermoactinomyces
vulgaris (NCBI accession no. 494645). B, the schematic
alignment of entire protease genes. The regions with sequence
similarity are indicated by black boxes. C, the
predicted transmembrane regions in cloned gene. The score of the
transmembrane region from outside to inside orientation
(o-i) is indicated by a solid line,
and the score in the reverse direction (i-o) is
denoted by a dotted line. The sequence region that has a
score higher than 500 is considered a transmembrane region.
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Fig. 4.
Identification of the 43-kDa protein as the
product of the cloned gene. Proteins from total cell extract of
A. pyrophilus (lane 1), cytosolic fraction
(lane 2), and cell wall fraction (lane 3) were
analyzed by Western blot (A) or by activity assay after
separation in SDS-PAGE containing 0.1% gelatin (B).
C, immunoprecipitation. Lane 4, control (no
addition of antibody); lane 5, control (addition of bovine
serum albumin instead of antibody); lane 6, addition of
antibody.
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Fig. 5.
Localization of Aquifex
protease. A, localization of Aquifex
protease. The samples were fractionated by ultracentrifugation.
Lane 1, cytosolic fraction; lane 2, membrane
fraction; lane 3, cell debris. B, extraction of
Aquifex protease from the membrane fraction with 1 M NaCl or 0.1% Triton X-100. After incubation for 1 h
on ice, insoluble membranes were collected by centrifugation at
100,000 × g for 1 h. Lane 4, insoluble
membrane fraction after treatment with 1 M NaCl; lane
5, soluble fraction after treatment with 1 M NaCl;
lane 6, insoluble membrane fraction after treatment with
0.1% Triton X-100; lane 7, soluble fraction after treatment
with 0.1% Triton X-100.
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Fig. 6.
Western analysis of the expressed protein in
E. coli. A, Coomassie-stained PAGE.
B, Western analysis in SDS-PAGE containing 0.1% gelatin.
M, molecular mass markers; lane 1, crude extract
of E. coli harboring pET 21a; lane 2, crude
extract of E. coli harboring the expression vector before
induction; lane 3, crude extract after induction with 1 mM isopropyl-1-thio- -D-galactopyranoside;
lane 4, crude extract after heat treatment at 95 °C for
20 min.
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Fig. 7.
Characteristics of the protease activity from
protein extract of the cell wall fraction of A. pyrophilus. A, the effect of temperature.
B, the effect of pH. After the same amount of protein was
separated in SDS-PAGE containing 0.1% gelatin, each lane
was incubated at different temperatures or pH values for 30 min.
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Fig. 8.
Characteristics of the protease expressed in
E. coli. The partially purified protease was assayed
for its protease activity using azocasein as a substrate at different
pH values at 85 °C (A) or different incubation
temperatures at pH 8.0 (B) for 30 min. Sodium phosphate
buffer (100 mM) was used at pH 6-7 ( ), 100 mM Tris-HCl buffer at pH 7-9 (
), and 100 mM
CAPS buffer at pH 9-11 (
).
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Fig. 9.
Thermostability of the expressed
protease. Activity assay of the partially purified protease was
performed using azocasein. The protein sample was incubated at 85 (A) or 105 °C (B) for the indicated time, and
the residual activity was measured.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
Comparison of amino acid composition of Aquifex protease with those of
several serine proteases
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Science and Technology, Korea and by United States Department of Energy Grant DE-AC03-76SF0098.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Structural Biology
Center, Korea Inst. of Science and Technology, P.O. Box 131, Cheongryang, Seoul, 136-791 Korea. Tel.: 82-2-958-5936; Fax:
82-2-958-5939; E-mail: ygy{at}kistmail.kist.re.kr.
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
2 I.-G. Choi, W.-G. Bang, S.-H. Kim, and Y. G. Yu, unpublished results.
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