From the
A novel property of the bacterial outer membrane protein T,
OmpT, has been discovered. It is active under extreme denaturing
conditions. This finding emerged during characterization of a protease
associated with the degradation of recombinant proteins expressed as
inclusion bodies in Escherichia coli. These inclusion body
proteins are stable to proteolytic degradation until they are
solubilized by denaturation. The protease that degrades them under
denaturing conditions was identified as OmpT on the basis of substrate
specificity, inhibitor profile, and confirmation that its N-terminal
sequence is identical with that of OmpT. A previously unknown property
of this enzyme, OmpT's preference for denatured substrates, may
provide a clue to its physiological function. To facilitate further
characterization of this proteolytic activity, we have optimized a
system to extract and assay OmpT under denaturing conditions using a
soluble substrate, rabbit muscle creatine kinase.
The OmpT protease,
Other possible roles for OmpT may be
hypothesized from analogy to other proteases. There are functional and
structural similarities between OmpT and the plasminogen activator of
Yersinia pestis (47% sequence identity) and OmpT is able to
cleave plasminogen to plasmin (9). Since the plasminogen activator is a
virulence factor, it has also been suggested that OmpT may contribute
to the pathogenic potential of E. coli(10) .
OmpT is
known to create problems in the expression of recombinant proteins.
Several recombinant proteins expressed in the cytosol have been
proteolyzed by OmpT after cell
lysis
(1, 8, 9, 11, 12, 13, 14) .
As mentioned above, OmpT also interferes with recovery of expressed
proteins by degrading recombinant fusion proteins secreted into the
periplasmic space
(6) . Our results identify OmpT as the protease
involved in the degradation of denatured inclusion body proteins, and
this has led to the discovery of a previously unknown property of the
OmpT protease: activity under extreme denaturing conditions.
In order to characterize
this protease activity in a soluble system, we optimized the assay
reported earlier
(15) . As shown in Fig. 1, TCK inclusion
bodies under native conditions were not degraded (lanes 1 and
3), but were degraded extensively after incubation with 8
M urea (lane 2). There was no significant degradation
of denatured TCK inclusion bodies after the protease was extracted
(lane 4). The protease activity that is removed by detergent
extraction contains a mixture of proteins as shown in lane 5.
It can be incubated with soluble RMCK, under denaturing conditions, to
give a similar degradation pattern (lane 7) to the denatured
TCK inclusion bodies (lane 2). The RMCK is not proteolyzed
when denaturant is left out of the assay mix (lane 6). These
data show that soluble RMCK incubated with detergent-extracted protease
can be used in place of inclusion body TCK to assay for the proteolytic
activity. This result is not surprising as TCK and RMCK are very
similar proteins (85% sequence identity).
The protease extract isolated from TCK
inclusion bodies has a substrate specificity for the scissile bond
between paired basic residues, the same specificity as that previously
reported for OmpT
(2) (Fig. 3).
Using the RMCK assay, the
degradation pattern for the protease extracted from TCK inclusion
bodies (Fig. 4, lane 2) is essentially the same as that
of the overexpressed OmpT extract (Fig. 4, lane 4). In
contrast, the detergent extract from a membrane preparation of the OmpT
deletion mutant has no proteolytic activity using our assay conditions
(Fig. 4, lane 6).
We have found the E. coli protease OmpT to be
responsible for the degradation of TCK inclusion bodies under extreme
denaturing conditions required for the solubilization of inclusion body
TCK. The identification of this protease as OmpT was based on four
criteria: substrate specificity, inhibitor profile, the complete lack
of proteolytic activity of an OmpT
Technical
difficulties in extraction and purification of the protease from
inclusion bodies resulted in incomplete purification, preventing us
from obtaining direct confirmation of its identity. The major
contaminants were identified as the E. coli porin proteins,
OmpF and OmpC. The physical nature of inclusion bodies has not been
explored, and little is known either about the forces which hold
inclusion bodies together or exactly how they are formed. Although the
scheme used to purify the protease was quite similar to that reported
for OmpT, the extraction conditions required to remove the protease
from the TCK inclusion bodies required high detergent concentrations
(2.5%) and was much more rigorous than that required for the removal of
OmpT from the membrane. This resulted in extraction of other integral
membrane proteins not present in the reported descriptions of OmpT
purification
(2) and led to increased difficulty in purifying
the protease activity from the inclusion body extract. Even though this
resulted in an incomplete purification, the enrichment of the 37-kDa
band verified as OmpT by N-terminal sequence determination shows that
OmpT was present in relatively high concentration in the most active
fraction.
The fact that OmpT is active under extreme denaturing
conditions has implications for elucidating the currently unknown
function of this enzyme. Although no native substrates for OmpT have
been found, its location in the outer membrane combined with its
unusual and selective substrate specificity and the preference for
denatured substrates lends support to a suggested role in processing
proteins secreted through the outer membrane
(7, 8) .
This raises the question of whether OmpT has a functional homolog in
eukaryotic systems. We find no evidence, however, for an ancestral link
between OmpT and eukaryotic processing proteins such as kex2 in yeast,
a unicellular eukaryote
(22) . Thus, even though the substrate
specificity of OmpT is similar to that of kex2 (the scissile bond is on
the carboxyl side of two consecutive basic residues), their inhibitor
profiles are markedly different. kex2 is sensitive to thiol modifying
reagents, whereas OmpT is not inhibited by thiol modifying reagents,
but rather is sensitive to the serine protease inhibitor
diisopropylfluorophosphate
(2) . In addition, there appears to be
no sequence similarity between kex2 and OmpT.
The preference of OmpT
for denatured substrate as observed in our system is an interesting and
as yet not well understood property of the enzyme. This finding may
help to illuminate other observations of OmpT degradation of regions of
recombinant proteins expressed in E. coli which appear to
contain relatively unorganized structures in the native protein. For
example, a conformational study of recombinant human
The preference of OmpT for
denatured substrates, its limited substrate specificity, and its
activity under denaturing conditions make OmpT particularly well suited
for specific and selective modification of proteins not only in
vivo but in vitro as well. Trypsin, which is also active
in 4 M urea
(26) , is most commonly used for this
purpose, although the substrate specificity is broader. The narrower
substrate specificity of OmpT suggests its potential for use in
obtaining internal sequence of proteins without excessive degradation.
In addition, OmpT appears to be a highly efficient enzyme under
denaturing conditions. Ten µg of the unpurified extract can degrade
100 µg of CK at 37 °C in 30 min to give reproducible
degradation patterns. Work to develop this enzyme as a biochemical tool
is on-going.
The ability to proteolyze substrates at high denaturant
concentrations is not a property unique to OmpT. Some other proteases
which retain activity in 8 M urea are subtilisin
(27) ,
papain (28), thermophilic aminopeptidase I
(29) , extracellular
protease from Penicillium notatum(30) ,
thermolysin
(31) , and Aeromonas aminopeptidase
(32) . It would be interesting to determine
whether the stability of OmpT can be correlated with structural
properties similar to those exhibited by some of these other enzymes.
Our conclusion that the protease responsible for the degradation of
inclusion body CK in the presence of denaturant is OmpT may be useful
in resolving problems associated with the recovery of active proteins
expressed as inclusion bodies in E. coli. For example, the
BL21 strain of E. coli, used with a range of commercially
available expression vectors, is OmpT
Inhibitor concentrations for the protease degradation
assay were determined by the amount of inhibitor required to block any
degradation of RMCK at that concentration. The inhibitors that
completely block the protease activity at 5 mM are labeled
+ and at 0.5 mM ++. EDTA and
phenylmethylsulfonyl fluoride did not inhibit the protease activity at
or below 12.5 mM.
We thank Dr. James W. Bodley for critical comments
and helpful discussions and Dr. George Georgiou for the plasmid pML19
and the E. coli strain UT5600.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
located in the outer
membrane of Escherichia coli, has a narrow substrate
specificity, primarily for paired basic residues
(1, 2) .
OmpT expression is thermoregulated showing much lower expression levels
below 32 °C
(3) . Although OmpT has been purified
(4) and the ompT gene cloned and sequenced
(5) ,
little is known about the in vivo function of the enzyme. The
ompT gene is apparently not essential as its deletion has no
effect on growth rate of E. coli(6) . Although no
natural substrates for OmpT have been identified, observations of
activity on recombinant proteins expressed in E. coli are
suggestive of possible physiological functions. For example, a fusion
protein carrying the HlyA transport signal, which was expressed in
E. coli and secreted through the outer membrane, was observed
to be cleaved by OmpT (7). OmpT cleaves another fusion protein
including both the C-terminal domain of the Neisseria Iga
protease precursor and the Vibrio cholerae toxin B subunit (CtxB), causing the extracellular release of the
toxin B subunit
(8) . In addition, OmpT appears to be involved in
the degradation of a
-lactamase-protein A fusion protein in the
periplasmic space of E. coli(6) . These observations
point to a possible role for OmpT in the processing of precursor
polypeptides and suggest that it may also have a role in
turnover/degradation of membrane proteins of both the inner and outer
membranes of E. coli.
Bacterial Strains and Growth
Conditions
Torpedo californica creatine kinase (TCK)
was expressed as insoluble inclusion bodies in the E. coli strain JA221 as described previously
(15) . OmpT was
overexpressed in the E. coli strain UT5600 (an OmpT deletion
mutant) transformed with the plasmid pML19
(1) containing the
ompT gene (a generous gift from Professor George Georgiou).
UT5600 cells and overexpressed OmpT (UT5600 harboring pML19) were grown
to an A of 2.0 in LB at 37 °C.
Extraction of the Protease Activity from TCK Inclusion
Bodies
The TCK inclusion bodies were resuspended at
approximately 0.5 mg/ml TCK in 50 mM Tris, pH 8.5, containing
2.5% octyl glucoside (Sigma) and 5% -mercaptoethanol. This
suspension was incubated with slow shaking at 37 °C for >16 h
and centrifuged at 12,000
g for 30 min. Purification
of inclusion body proteins was evaluated by SDS-PAGE. The extract was
exchanged by ultrafiltration into 1% octyl glucoside, 50 mM
Tris, pH 8.5. Protein concentration was determined by the bicinchoninic
acid (BCA) protein concentration assay (Pierce), using bovine serum
albumin as a standard.
Protease Degradation Assay of Inclusion Bodies
TCK
inclusion body protein was resuspended to 1 mg/ml in 50
mM Tris, pH 8.5. Thirty microliters of this suspension was
centrifuged for 10 min at 12,000
g, and the
supernatant was discarded. The protein pellet was resuspended to a
concentration of 2 mg/ml in 50 mM Tris, pH 8.5. Solid
ultrapure urea (U.S. Biochemical Corp.) and 1 M DTT were added
to give final concentrations of 8 M and 100 mM,
respectively, when the total volume was adjusted to 30 µl (1 mg/ml
protein). The extracted and unextracted TCK inclusion bodies were
assayed for protease activity under either native (DTT only) or
denaturing (DTT + urea) conditions by incubation at 37 °C for
30 min. Degradation products were analyzed by SDS-PAGE .
RMCK Degradation Assay Using Protease
Extract
Rabbit muscle creatine kinase (RMCK) was used as a
soluble substrate for the protease. The reaction contained: RMCK (1
mg/ml), protease extract (0.1 mg/ml protease extract), DTT (1
mM), urea (4 M, for denatured samples only), and
0.15% octyl glucoside in 50 mM Tris, pH 8.5, in a final assay
volume of 100 µl. Assay conditions were identical with those used
for inclusion bodies. Inhibition experiments were done using the
soluble RMCK assay by adding potential inhibitors to a final
concentration of 0.5, 1, 5, 10, or 12.5 mM to the assay
mixture immediately prior to adding the protease extract. The degree of
protease inhibition was assumed to correlate with the amount of
full-length RMCK remaining as judged by SDS-PAGE.
Determination of the Urea Optimum of the Protease
Extract
Residual CK activity was measured following refolding of
RMCK
(15) denatured at various urea concentrations (0-8
M) in 50 mM Tris, pH 8.5. CK activity was determined
by the method of Tanzer and Gilvarg
(16) .
Preparation of Membrane Extracts
OmpT deletion
mutant strain UT5600 cells and UT5600 transformed with plasmid pML19
(overexpressing OmpT)
(5) cells, grown as described, were
resuspended in 50 ml of 50 mM Tris, pH 8.5, with 0.5 mg/ml
lysozyme, incubated overnight at 4 °C, and sonicated. The cell
lysate was centrifuged at 20,000 rpm for 30 min, and the insoluble
material was extracted as described.
Enzyme Purification
JA221 cells expressing TCK
(15.5 g) were lysed, and the protease activity was
extracted
(17) . The extract was exchanged into 1% octyl
glucoside, 50 mM Tris, pH 8.0 (buffer A) by ultrafiltration.
Protease extract (27 mg) was filtered (0.2 µM) and loaded
onto an AP-1 Waters column (10 100 mm) containing benzamidine
Sepharose (Pharmacia Biotech Inc.). The column was washed with 40 ml of
buffer A. The protease activity was eluted using a linear gradient of
buffer B (buffer B contains 1 M NaCl + buffer A) in one
peak at 200 mM NaCl, 50 mM Tris, pH 8.0. The active
fraction contained 9.5 mg of protein and was exchanged back into buffer
A. The benzamidine-purified extract was then loaded onto a HR(5/10)
Mono Q anion exchange column (Pharmacia). The column was washed with 20
ml of buffer A, and the protease activity was eluted using a linear
NaCl gradient (buffer B) and eluted from the column in 4 incompletely
resolved peaks from 180 mM NaCl to 240 mM NaCl. The
active fractions from Mono Q contained 3 mg of protein.
N-terminal Sequence Determination of the Protease
Extracted from Inclusion Bodies
The most active fraction from
the Mono Q anion exchange column was fractionated by SDS-PAGE,
electroblotted onto a Problot membrane, and stained with Coomassie Blue
R-250. The protein in the enriched 37-kDa band was subjected to 8
rounds of automated Edman degradation at the UCSF Biomolecular Resource
Center.
Proteolysis Occurs under Denaturing
Conditions
Formation of inclusion bodies is a major problem
encountered in bacterial expression systems. During attempts to recover
active enzyme by denaturation and refolding, we have observed
proteolytic degradation in inclusion bodies containing both T.
californica creatine kinase (TCK) and bovine pancreatic trypsin
inhibitor
(15, 17, 18) . As reported previously,
this proteolysis occurs under the extreme denaturing conditions (8
M urea) which are necessary to solubilize inclusion body
proteins prior to refolding. The proteolytic activity can be isolated
by detergent extraction of the insoluble pellet containing the TCK
inclusion bodies and membrane fragments.
Figure 1:
SDS-PAGE (12.5%) comparison of protease
activity in TCK inclusion bodies and in a soluble assay system.
Lane 1, TCK inclusion bodies under native conditions; lane
2, under denaturing conditions (8 M urea). Lane
3, TCK inclusion bodies after the removal of proteolytic activity
under native conditions; lane 4, under denaturing conditions.
Lane 5, the protease extract (concentrated 100-fold over
amounts used for the degradation assays shown in lanes 6 and
7). Lane 6, the detergent extract containing the
proteolytic activity was incubated with soluble RMCK under native
conditions; lane 7, under denaturing conditions (4 M
urea).
Optimum Denaturant Concentration for
Proteolysis
The optimum denaturant concentration for protease
activity was determined by both the SDS-PAGE assay and by quantitation
of residual CK activity following refolding of RMCK that had been
denatured in the presence of the protease extract. Urea was used in
preference to guanidine HCl because it can be used in a gel assay, and
care was taken to use only freshly prepared, ultrapure urea in the
experiments. The urea optimum for the proteolytic activity on substrate
RMCK was 4-5 M urea (Fig. 2). Although the
degradation of RMCK (data not shown) also correlated with the loss of
residual CK activity following refolding, residual CK activity was a
more quantitative indicator of the protease activity under a range of
denaturant concentrations than the gel assay.
Figure 2:
Denaturant optimum for protease activity
on substrate RMCK. Loss of CK activity (units/ml) following refolding
was measured at increasing concentrations of
urea.
Identification of the Protease as
OmpT
Characterization of this protease allowed us to ask whether
this activity could be attributed to any known bacterial protease. Our
experimental evidence suggests that the E. coli protease OmpT
is responsible for the proteolytic degradation of TCK inclusion bodies
under the denaturing conditions required for solubilization and
refolding. This conclusion is based on several types of experimental
evidence as detailed below.
Figure 3:
Substrate specificity of protease activity
on denatured TCK inclusion bodies (top sequence) and denatured
soluble RMCK (bottom sequence). Alignment of TCK and RMCK
shows locations in the sequences where proteolysis occurs. Degradation
products of both enzymes were fractionated by SDS-PAGE, and N-terminal
sequences were determined for the major degradation fragments
(boxed). Arrows mark the position of the scissile
bond in each sequence.
The protease extract
also has the same inhibitor profile as that reported for OmpT
(2) (). Benzamidine, ZnCl, and
CuCl
inhibit proteolysis of substrate RMCK at millimolar
concentrations as previously reported for OmpT
(2) , whereas EDTA
and phenylmethylsulfonyl fluoride do not.
Figure 4:
SDS-PAGE (12.5%) comparison of protease
activity from membrane extracts of JA221 cells (used to express TCK),
UT5600 (OmpT), and UT5600 + pML19 (overexpressed
OmpT). Lane 1, substrate RMCK incubated with membrane
detergent extracts from E. coli JA221 cells used to express
TCK under native conditions; lane 2, under denaturing
conditions (4 M urea). Lane 3, substrate RMCK
incubated with an extract from UT5600 cells with overexpressed OmpT
(pML19) under native conditions; lane 4, under denaturing
conditions (4 M urea). Lane 5, substrate RMCK
incubated with an extract from UT5600 cells under native conditions;
lane 6, under denaturing
conditions.
A comparison of membrane extracts
from cells overexpressing OmpT and cells that do not express OmpT shows
a large band of about 37 kDa present in the overexpressed OmpT extract
(Fig. 5, lane 2) and absent in the UT5600 extract (no
OmpT expressed) (Fig. 5, lane 3). The purification of
the protease extracted from TCK inclusion bodies is also shown in
Fig. 5
(lanes 4, 5, and 6). This
extract also contains a 37-kDa band that increases in intensity at each
step of the protease purification. The N-terminal sequence of this
enriched band is STETLSFT, which is identical with that of the mature
OmpT protease
(5) . N-terminal sequence analysis of the large
band directly below the band identified as OmpT (Fig. 5, lane
6) suggests that they are the porin proteins, OmpF and OmpC. This
band was blotted and subjected to 6 rounds of Edman degradation
resulting in a primary and a secondary sequence. The primary sequence
is AEIYNK, which is the N-terminal sequence for the mature OmpF porin
protein
(19, 20) . The secondary sequence that was
obtained from this band, AEVYNK, corresponds to the N-terminal sequence
of the mature OmpC porin protein
(21) .
Figure 5:
Purification of the protease activity
extracted from TCK inclusion bodies compared to membrane extracts of
OmpT and overexpressed OmpT. Lane 1,
molecular mass marker; lane 2, membrane extract from
overexpressed OmpT (UT45600 + pML19); lane 3, membrane
extract from OmpT
(UT5600). The purification of the
protease extract from TCK inclusion bodies: lane 4, unpurified
protease extract; lane 5, benzamine Sepharose fraction; and
lane 6, Mono Q anion exchange
fraction.
mutant, and
N-terminal sequence confirmation that the 37-kDa band enriched by
purification of the protease activity is identical with the N-terminal
sequence of OmpT and corresponds to its predicted molecular mass. The
overexpressed OmpT also appears to have the same degradation pattern as
the protease activity extracted from TCK inclusion bodies.
-interferon
shows that the 20 amino acids at the C terminus lacks structure in
comparison to the rest of the molecule. This was the only region of the
protein susceptible to proteolysis under nondenaturing
conditions
(2) . When
-helix-breaking amino acids were
introduced into a linker sequence of the fusion protein containing PhoA
and hemolysin A expressed in E. coli, the cleavage of OmpT
sites in the linker sequence was substantially increased
(7) .
One likely explanation for these results is that OmpT acts on
relatively unstructured regions of the substrate, mimicked in our
soluble assay system by addition of denaturant. At high denaturant
concentrations (above 5 M urea), there is reduced activity of
OmpT on substrate RMCK. As the urea concentration increases, it is
likely that the protease itself begins to denature, leading to a loss
of activity. This suggests that the optimum denaturant concentration
for OmpT proteolysis will be substrate-dependent. The efficiency of
proteolysis will involve a trade-off between substrate stability and
OmpT stability at a given concentration of denaturant. (Creatine kinase
shows loss of activity and denaturation of tertiary structure at urea
concentrations below those used in our assay (23-25).) Under our
assay conditions, there is presumably sufficient denaturant present to
induce a relaxed creatine kinase structure susceptible to proteolysis.
The concentration of urea required to achieve this is, however,
insufficient to denature OmpT itself.
. In cases where
proteolysis of inclusion body proteins occurs during denaturation and
refolding, use of such a strain might provide a simple and viable
solution. Alternatively, use of OmpT inhibitors may be useful for
recovery of inclusion body proteins from such systems. Although the
generality of this proteolysis phenomenon has not been formally
established, two recombinant proteins (TCK and bovine pancreatic
trypsin inhibitor) have been shown to be proteolyzed during
denaturation and refolding from inclusion bodies
(15) . We
suggest that this protease may be responsible for the proteolysis of
other inclusion body proteins degraded during the denaturation and
refolding procedures used to recover soluble protein from such systems.
Many schemes for recovery of inclusion body proteins include extraction
procedures for removal of contaminating proteases prior to
denaturation. It is possible that OmpT proteolysis of inclusion body
proteins could be minimized by such procedures as Triton X-100 washes.
As reported previously, however, Triton treatment may only be effective
with OmpT when it is used in the lysis buffer
(17) . In contrast
to octyl glucoside, extensive Triton or Triton/deoxycholate washes of
the inclusion body CK after the cells were lysed did not extract the
protease effectively.
(
)
Table:
Inhibition of the protease activity on denatured
substrate RMCK was measured by loss of full-length RMCK on 12.5%
SDS-PAGE gels
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.