©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Activity of OmpT
PROTEOLYSIS UNDER EXTREME DENATURING CONDITIONS (*)

Camille Bodley White (1), Qun Chen (2), George L. Kenyon (1), Patricia Clement Babbitt (1)(§)

From the (1) Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 and the (2) Department of Food Science, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The OmpT protease,() 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.

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.


MATERIALS AND METHODS

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.


RESULTS

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.

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).


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.

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).


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.

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).


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.




DISCUSSION

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 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.

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 -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.

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. 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

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.



FOOTNOTES

*
This work was supported by United States Public Health Service Grant AR17323. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: School of Pharmacy, University of California, Box 0446, San Francisco, CA 94143. Tel.: 415-476-3784; Fax: 415-476-0688.

The abbreviations used are: OmpT, outer membrane protein T; RMCK, rabbit muscle creatine kinase; TCK, Torpedo californica creatine kinase; PAGE, polyacrylamide gel electrophoresis; BCA, bicinchoninic acid; DTT, dithiothreitol.

P. C. Babbitt, unpublished results.


ACKNOWLEDGEMENTS

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.


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