Purification of Integral Outer-Membrane Protein OmpC, a Surface Antigen from Salmonella typhi for Structure–Function Studies: A Method Applicable to Enterobacterial Major Outer-Membrane Protein

Analytical Biochemistry 283, 64 –70 (2000) doi:10.1006/abio.2000.4634, available online at http://www.idealibrary.com on

Purification of Integral Outer-Membrane Protein OmpC, a Surface Antigen from Salmonella typhi for Structure– Function Studies: A Method Applicable to Enterobacterial Major Outer-Membrane Protein A. Arockiasamy and S. Krishnaswamy 1 Bioinformatics Centre, School of Biotechnology, Madurai Kamaraj University, Madurai-625 021, India

Received December 29, 1999

Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDSPAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure–function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins. © 2000 Academic Press Key Words: outer membrane; OmpC; porin; purification; Salmonella typhi.

Gram-negative bacterial porins play a major role in the physiology of the bacterium, by allowing small hydrophilic molecules to pass through the channel (1). Their expression was preferential according to the en1 To whom correspondence should be addressed. Fax: ⫹91-452859105. E-mail: [email protected].

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vironmental conditions of the bacterial growth (2, 3). The outer-membrane protein profile is altered when enterobacteria encounter adverse conditions, during infection and survival in the host background. This physiological response could be important for survival in such environments (4). Salmonella typhi is an obligatory human pathogen and causes typhoid fever, which continues to be a major health problem in developing countries (5). In this context, the involvement of outermembrane proteins (OMPs) 2 of Salmonella in eliciting a protective immune response has been demonstrated (6, 7). The possibility of S. typhi OmpC, a homotrimer with a monomer of 357 amino acids and MW 39 kDa, being expressed throughout the infection period has also been emphasized (8). S. typhi OmpC has been shown to display heterologous epitopes on the cell surface (9). Since OmpC is the major surface antigen with unique surface-exposed epitopes, as evidenced by Salmonella porin-specific monoclonal antibodies (10), it could be used in diagnosis and multivalent vaccine design. Hence, S. typhi OmpC is a good candidate antigen for structural studies related to immunology. Earlier we have predicted the sequential epitopic regions of S. typhi OmpC based on sequence alignment (11). Prediction was improved using a homology-based model (PDB: 1IIV) built for this protein. S. typhi OmpC was crystallized (12) in order to determine the structure and to characterize the antigenic regions in terms of their uniqueness at the structural level. Porin needs to be purified routinely, in large quantities, for structure–function studies using biophysical methods. Purification of individual outer-membrane proteins from wild-type strains has not been successful (13). Re2 Abbreviations used: OMPs, outer-membrane proteins; LPS, lipopolysaccharides; ␤-ME, ␤-mercaptoethanol.

0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

PURIFICATION OF ENTEROBACTERIAL MAJOR OUTER-MEMBRANE PROTEIN

ported porin-extraction methods have resulted in more than one protein from the membrane preparation and none of them has yielded a single major OMP without further purification (14 –16). Moreover, many of these methods use mutants as a strategy to get rid of unwanted contaminating OMPs or, otherwise, have used recombinant bacteria expressing the porin of interest. Purification of E. coli PhoE using a His-tag construct has been reported (17). However, the tag seems to have reduced the trimer stability and its possible use in crystallization of active trimer is questionable. Here we report a method for rapid isolation of the major OMP, OmpC, from the crude membrane preparation from S. typhi Ty21a and show that this method was successfully used to isolate OmpC from E. coli, grown under high osmolarity conditions, also. The applicability of this method for major OMP preparation from other gram-negative bacteria is discussed. MATERIALS AND METHODS

Strain and Bacterial Culture Professor V. R. Muthukkaruppan, Department of Immunology, Madurai Kamaraj University, kindly provided S. typhi Ty21a (18). This strain is useful as an oral vaccine for typhoid. It is a galactose epimerase mutant (galE), producing incomplete lipopolysaccharides (LPS). The Genetic Engineering Research Unit, School of Biotechnology, Madurai Kamaraj University, provided the strain E. coli HB101. S. typhi Ty21a was grown in a media containing 1% beef extract, 1% peptone, and 0.5% NaCl for 14 h in an incubator shaker set at 37°C and 200 rpm. E. coli HB101 was grown in Luria broth (1.0% tryptone, 0.5% yeast extract, and 1.0% NaCl) for 12 h under the same conditions. Cells from large-scale cultures (4 L) were centrifuged at 7000 rpm for 30 min at 4°C (rotor RPR-12-2, Hitachi highspeed centrifuge). The pellet was washed twice with 0.85% saline and centrifuged at 7000 rpm for 30 min to get the final pellet which was stored overnight at ⫺20°C before further use. A typical culture of 4 L yielded about 14 –17 and 11–12 g wet weight of cell pellets of S. typhi Ty21a and E. coli HB101, respectively. Preparation of Crude Membrane The cell pellet stored at 20°C was thawed at 4°C, suspended in Tris-HCl (pH 7.2) and sonicated (150 W) using a Labsonic System (Lab-line Instruments, U.S.A.). The cell suspension was passed thrice through the continuous sonication chamber each time for 15 or 10 min for Ty21a and HB101, respectively, with a 5- to 10-min gap. The cell sonicate was centrifuged at 11,000 rpm (rotor RPR20-2) for 10 min at 4°C to remove the undisrupted cells. The supernatant was centrifuged at

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30,000 rpm for 90 min at 20°C using a Hitachi ultracentrifuge (rotor RP-50T). The pellet containing the crude membrane was used for further processing. Major OMP Extraction The salt-extraction procedure reported earlier for porin isolation (19) was modified to prepare the major OMP in a (i) small and (ii) large scale. Buffers used for the extraction and purifications were freshly prepared. (i) Crude membrane pellet collected from a 1-L culture was thoroughly suspended in 25 ml of Buffer I (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2, and 2% SDS) and incubated at 37°C for 18 h in a water bath. The suspension was centrifuged at 12,000 rpm for 30 min at 20°C and the pellet was collected. This pellet was resuspended in 7 ml of Buffer II (50 mM Tris-HCl, 10 mM MgCl 2, 5 mM EDTA, 1% SDS, and 0.05% ␤-ME—added just before use). This suspension was incubated at 37°C for 2 h and centrifuged at 12,000 rpm for 30 min at 20°C. Pellet from the above step was again resuspended in 3 ml of Buffer III (50 mM Tris-HCl, 5 mM EDTA, 0.2% SDS, 0.4 M NaCl, and 0.05% ␤-ME) and incubated for 2 h at 37°C. The suspension was then centrifuged at 12,000 rpm for 20 min at 20°C. The supernatant, rich in porin, was collected. The above step was repeated at least twice, each time with 2 ml of Buffer III. The collected supernatants, containing crude porin, were pooled after the protein profile was checked using SDS-PAGE. All extraction buffers had 3 mM NaN 3 to avoid bacterial contamination. (ii) Large-scale extractions were done using cells from 4-L cultures. The same method was used with 100 ml of Buffer I, 28 ml Buffer II, and 9 ml of Buffer III. Extraction with Buffer III was found to have porin up to six repeated extractions. The extracts were stored at room temperature for further use. Chromatography and Ultrafiltration The major OMP extracts were subjected to chromatographic and ultrafiltration methods to remove the free as well as loosely bound LPS. The pooled sample of crude porin extracts from S. typhi Ty21a cells (3 ⫻ 4-L cultures) was concentrated using an Amicon Stirred cell (Millipore) with YM-10 or YM-50 membrane. The protein sample (45 mg in 3 ml) was passed through the gel-filtration column (i.d., 16 mm; bed height, 98 cm; bed volume, ⬃197 ml; void volume, ⬃72 ml; matrix, Sephacryl S-200 HR Pharmacia Biotech; flow rate, 7–10 ml/h; fraction size, 1 ml). Extraction buffer III was used for equilibration and elution. For anion-exchange chromatography, porin in buffer III was exchanged to a buffer containing 50 mM Tris-HCl (pH 7.7), 1% octyl-POE (Bachem), and 3 mM NaN 3 using the stirred cell with a 50 kDa cutoff membrane. A total

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of about 90 mg of porin (from 5 ⫻ 4-L cultures) was then applied (18 –19 ml/h) onto a Q-Sepharose fast-flow (Sigma) column (15 mm i.d., 15-ml volume, Spectrum) packed with 10 ml of media. The column was preequilibrated with about 100 ml of 50 mM Tris-HCl (pH 7.7) and followed by 50 ml of the buffer used for exchange. The flowthrough of the applied sample was passed through the column 7 times, to allow for maximum binding. Then the column was washed with the equilibration buffer and the wash was collected to check with OD 280 and SDS-PAGE. Continuous gradient elution was done with the above buffer containing 150 – 450 mM NaCl. Fractions (0.5 ml) were collected at the flow rate of ⬃18 ml/h and checked for protein using OD 280 measurements. Peak fractions were pooled, after the purity was checked on SDS-PAGE, and concentrated before storage. Protein was estimated using a modified Lowry method (20) with BSA as a standard. SDS-PAGE with silver staining was used to monitor the LPS removal. SDS-Polyacrylamide Gel Electrophoresis The major OMP extracts and column-purified porin samples were analyzed by SDS-PAGE with or without 8 M urea. Protein samples were mixed with sample buffer and applied onto 10, 11, or 13% polyacrylamide SDS slab gels. The sample buffer also had 8 M urea, in case of urea SDS-PAGE. Samples were loaded as either unboiled, to detect the oligomer, or boiled for 5 min at 100°C to dissociate the trimers into monomers, and electrophoresed at constant voltage in the discontinuous buffer system (21). The gels were stained with either silver nitrate or Coomassie blue (R-250). N-terminal sequencing of the S. typhi Ty21a porin transferred on to PVDF membrane was done (courtesy of Dr. Dinakar M. Salunke) at the National Institute of Immunology, New Delhi, India. RESULTS AND DISCUSSION

Culture Conditions Growth conditions like pH and osmolarity have been shown to influence the outer-membrane protein production in gram-negative bacteria. The ompC and ompF genes, which code for the major OMPs in enterobacteria, are under a two-component regulatory system. The high osmolarity laboratory culture conditions used here were expected to induce OmpC production rather than OmpF, the major OMP under low osmolarity conditions. Unlike E. coli OmpC expression, which is less in media under low osmolarity conditions, S. typhi OmpC was shown to be expressed more (major OMP) under both low and high osmolarity conditions. Since S. typhi survives in the human circulatory system, where it encounters high osmolarity conditions, one can expect OmpC to be the major OMP in vivo also.

Preparation of Major OMP (OmpC) In order to standardize the method, several extractions were carried out with S. typhi Ty21a cells from 1-L cultures in the initial stages. The extracts were analyzed using SDS-PAGE and found to be reproducible in terms of the protein profile. The amounts of wet cells obtained in different batches were comparable (4 –5 g/L) and yielded porin in reproducible quantities (3.5–5 mg/L). This method was extended for large-scale preparation of porin using cells from 4-L cultures. The volume and concentration of buffers used to solubilize the pellets, in different steps of extraction, were kept constant irrespective of the variations in the pellet weight from batch to batch. The yield of porin from different batches was, however, comparable. This method is now routinely used in our laboratory to extract the major OMP from E. coli HB101 strain also. Since the cell pellet weight was considerably less in E. coli cultures, the pellet to buffer ratio in each step of porin extraction was modified based on the corresponding pellet weights from Ty21a, to increase the yield of porin. The yield of porin from E. coli is about 75 mg from a large-scale culture, which is three times more than that obtained from Ty21a. Incubation of crude membrane pellet suspension for 18 h in Buffer I at 37°C is the crucial step in eliminating all other proteins in the initial stages of extraction. OmpA and other OMPs seemed to be missing in the final extracts, unlike in other methods. This removal probably occurs during the 18-h incubation. This was evidenced from the Buffer I supernatant run on SDSPAGE (Fig. 2). The reason for this removal may be linked to the SDS solubilization. However, the rationale is not explainable by us. The purity of the major OMP in the porin extracts was achieved at the cost of some loss of major OMP in the first two steps as evidenced from the SDS-PAGE. Moreover, it was observed (22) that incubation of ultra-pellet suspension in Buffer I for longer times results in less LPS in the porin preparations. Purity The identification of outer-membrane proteins based on their mobility in SDS-PAGE was misleading due to anomalous mobility (23, 24). The mobility is dependent on various factors like association of different chemotypic LPS to the same protein molecule, solubilization temperature, and amount of bound SDS in samples that were boiled and unboiled (25, 26). The amount of ammonium persulfate in the separating gel also seems to affect the mobility (27). Though the role of ␤-mercaptoethanol is not yet understood, addition of this reducing reagent has been shown to alter the mobility of the outer-membrane proteins in the gel (28). A similar effect was observed with the Ty21a OmpC showing

PURIFICATION OF ENTEROBACTERIAL MAJOR OUTER-MEMBRANE PROTEIN

FIG. 1. SDS-PAGE 13% gel stained with Coomassie blue. Lanes: 1, standard marker proteins; 2, Ty21a cell lysate; 3, aliquot of supernatant after ultracentrifugation, which was discarded; 4, Buffer I supernatant; 5, Buffer II supernatant; 6 and 7, major OMP extract; 8 and 9, purified major OMP (OmpC); 10 and 11, E. coli HB101 major OMP extract. Samples were loaded either unboiled (lanes 6, 8, and 10) or after boiling for 5 min (1, 2, 3, 4, 5, 7, 9, and 11). All the samples, except in lanes 10 and 11, were from Ty21a.

differential intensity of the resolved bands from boiled samples and reduction of isoforms in unboiled samples. Purity of the S. typhi porin extract was analyzed based on the pattern in SDS-PAGE. Samples, both boiled and unboiled, in the presence of sample buffer, were loaded on to 13% gels stained with Coomassie blue (Fig. 1) or with silver nitrate (Fig. 2). The Buffer I supernatant loaded in lane 4 shows that most of the proteins along with other OMPs were removed during the 18-h incubation in Buffer I itself. The supernatant of Buffer II extraction was enriched in major OMP as seen in lane 5. The sample from pooled extracts loaded in lane 6, that was unboiled, migrated as a ladder containing closely moving thin bands while the respective purified porin loaded in lane 8 resolved into two bands. The ladder formation could be due to porin isoforms, as evidenced from earlier reports, which were absent or less in number in the purified porin oligomer. The presence of isoforms probably reflects the different chemotypic LPS bound to porin which could be reduced to a large extent on repeated cycles of buffer exchange using Amicon concentrators with 50-kDa cutoff membrane filters. Silver staining (Fig. 2) helped to identify the free LPS bands seen below the 25-kDa region. Coomassie-stained gels did not show these bands. But the porin extracted from wild-type E. coli HB101 showed only a single major OMP in both unboiled and boiled samples loaded on lanes 10 and 11, respectively, in Fig. 2. The column-purified porin, after extensive buffer exchange, which was unboiled did not show any free LPS (lane 8, Fig. 2). This indicates that the free

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and more of loosely bound LPS have been removed from the porin bands after column chromatography and extensive buffer exchange. Porin trimers, being stable in SDS, do not dissociate and migrate as trimeric oligomers when they were not heated to 100°C (unboiled). In most cases, boiling with sample buffer dissociates the trimer and gives rise to denatured porin with the mobility corresponding to the monomer molecular weight. The boiled samples of S. typhi Ty21a major OMP preparation migrated as three bands with the approximate molecular weights ⬃47, 37, and 36 kDa. The bands corresponding to 36 and 37 kDa in the 13% gel migrated together as a single band in ⬍10% gel. This is in contrast to its mobility as a single band when it was expressed in E. coli HB101 and extracted using this method. The type of LPS attached to porin could vary depending on the bacterial strain. Apart from the culture conditions, genetic background can also influence the LPS synthesis, which can result in alteration in the mobility. As S. typhi Ty21a is a galE mutant, which produces incomplete LPS, it is possible that the attachment of these LPS gives rise to anomalous mobilities of Ty21a OmpC on gel electrophoresis. All three bands migrated together as a single band on application of the boiled samples onto an 11% gel with 8 M urea (Fig. 3). The LPS ladder, which was detected in normal SDS-PAGE, could not be seen in 8 M urea SDS-PAGE with silver staining. N-terminal sequencing, done up to 20 amino acids, confirmed that the three bands in the boiled sample of Ty21a porin correspond to a single polypeptide which is also supported by a single band seen in the urea SDS-PAGE. Dot enzyme immunoassays (dot blot) using monoclonal an-

FIG. 2. SDS-PAGE 13% gel stained with silver nitrate. Lanes: 1, standard marker proteins; 2, Ty21a cell lysate; 3, aliquot of supernatant after ultracentrifugation; 4, Buffer I supernatant; 5, Buffer II supernatant; 6 and 7, major OMP extract; 8 and 9, purified major OMP (OmpC); 10 and 11, HB101 major OMP extract. Samples were loaded either unboiled (lanes 6, 8, and 10) or after boiling for 5 min (1, 2, 3, 4, 5, 7, 9, and 11). All the samples, except in lanes 10 and 11, were from Ty21a.

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FIG. 3. 8 M urea SDS-PAGE 11% gel stained with silver nitrate. Lanes 1 and 2 were loaded with Ty21a major OMP extract; lanes 3 and 4 contain purified OmpC (used for crystallization). Lanes 1 and 3: samples were loaded unboiled. Lanes 2 and 4: samples were boiled for 5 min before loading. The sample buffer also had 8 M urea.

tibodies specific for Salmonella porins with whole cells and crude and purified porin show that this method has yielded the native oligomeric porin (29). Porins are well known for their tight association with LPS. Bands corresponding to LPS in the boiled crude extracts were more intense when compared with the sample that was not boiled (Fig. 4). This indicates the release of bound LPS on boiling, which might be the cause for increased intensity in the bands corresponding to LPS. Differences in the LPS type between Ty21a and HB101 extracts were also seen clearly from this gel. Surprisingly the free and loosely bound LPS released on boiling and during electrophoresis were not prominently seen in Fig. 2. The only difference in the crude extracts is that samples loaded in the gel for Fig. 4 were run a day after the extraction, whereas the samples used in the gel for Fig. 2 were stored at room temperature for a few months. Nevertheless, no differences in the mobility of protein bands were seen between the two cases, when the gels were stained with either Coomassie blue or silver nitrate. There was no difference observed with the purified samples irrespective of the storage.

free and bound forms of LPS. Free and loosely bound LPS could be removed by passing through either a gel-filtration or an ion-exchange column. A single peak was seen in the elution profile of the gel-filtration column. The isoforms of Ty21a OmpC, corresponding to 36 and 37 kDa in the boiled sample, could be separated well from the ⬃47-kDa isoform, in the initial fractions of the peak, on ion-exchange columns. However, the later fractions enriched in ⬃47-kDa form contained the other two also. Few peak fractions of the gel-filtration column were free of this form. The band corresponding to ⬃47-kDa isoform either disappeared or had reduced intensity on repeated buffer exchange of pooled gelfiltration column fractions. Addition of increased amounts of ␤-mercaptoethanol to the porin extracts before running it on SDS-PAGE also gave a similar result. The column fractions containing porin were pooled, concentrated, and used for final buffer exchange before crystallization. Buffer exchange using Amicon ultrafiltration devices, with 50-kDa molecular weight cutoff membranes, removed the bound LPS largely. Complete removal of bound LPS was not pos-

Removal of LPS from Ty21a Extracts OMP preparations were always contaminated with varying amount of different chemotypic LPS, which varies from species to species and the amount depends on the extraction procedure used. The amount of free LPS varies with each batch of preparation. Homogeneity, in terms of contaminating proteins, is the major criterion in any protein-purification method. Getting LPS-free protein is more important, in the case of bacterial outer-membrane proteins, especially when the purpose is to use the purified protein for crystallographic and other structural studies. The removal of LPS becomes crucial before crystallization of outermembrane protein (30). The porin extracts had both

FIG. 4. SDS-PAGE 13% gel stained with silver nitrate. Lanes: 1 and 2, S. typhi Ty21a major OMP extract (unboiled and boiled); 3, E. coli HB101 major OMP extract (boiled).

PURIFICATION OF ENTEROBACTERIAL MAJOR OUTER-MEMBRANE PROTEIN

sible even after boiling the porin in the presence of SDS-PAGE sample buffer as seen with carbohydrate staining (data not shown). A similar observation was made in an earlier study (31) using LPS-specific monoclonal antibodies. However, crystallization trials with the purified porin yielded reproducibly single crystals of OmpC. Usefulness of the Method The major OMP extracts can be used for various immunochemical and biophysical studies wherein complete removal of LPS is not required. Use of SDS, a chaotrophic anionic detergent, helps to solubilize and subsequently remove most of the protein components from the membrane. Since all other proteins were removed completely in the second step of major OMP preparation from Ty21a, SDS can be replaced subsequently by nonionic detergents of interest, like C 12E 9, for functional studies. In the case of EDTA-sensitive porin trimers, like R. capsulatus porin, this method can be adopted by including MgCl 2 or other salts instead of EDTA. After a few cycles of buffer exchange, without going for chromatographic methods, the porin extracts were used for epitope-mapping studies using monoclonal antibodies (32). Unfolding and refolding of major OMP from Ty21a were monitored with circular dichroism spectroscopy with pure as well as LPS-containing preparations (29). These results suggest that this method is useful for extracting the major OMP for immunochemical and biophysical studies. ACKNOWLEDGMENTS We thank Professor K. Dharmalingam and Professor V. R. Muthukkaruppan for their support and encouragement; Eswara Kumar, Lakshmi Mundkur, and Nandakumar for their help and useful discussions. Most of the work was carried out using the GERU facilities, MKU. We thank Dr. P. Palanivelu, Professor K. Veluthambi, Professor V. Sekar, and Dr. Usha for their lab facilities. Use of CPMB facilities is acknowledged. We acknowledge the help of Umesh and Sundara Baalaji in porin extractions. A.A. thanks CSIR, Government of India, for the fellowship. The research grant (SP/SO/D32/97) from DST, Government of India, is duly acknowledged.

REFERENCES 1. Nikaido, H. (1994) Porins and specific diffusion channels in bacterial outer membranes. J. Biol. Chem. 269, 3905–3908. 2. Puente, J. L., Verdugo-Rodriguez, A., and Calva, E. (1991) Expression of Salmonella typhi and Escherichia coli OmpC is influenced differently by medium osmolarity: Dependence on Escherichia coli OmpR. Mol. Microbiol. 5, 1205–1210. 3. Contreras, I., Munoz, L., Toro, C. S., and Mora, G. C. (1995) Heterologus expression of Escherichia coli porin genes in Salmonella typhi Ty2: Regulation by medium osmolarity, temperature and oxygen availability. FEMS Microbiol. Lett. 133, 105–111. 4. Delcour, A. H. (1997) Function and modulation of bacterial porins: Insights from electrophysiology. FEMS Microbiol. Lett. 151, 115–123.

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5. Pang, T., Levine, M. M., Ivanoff, B., Wain, J., and Finlay, B. B. (1998) Typhoid fever—Important issues remain. Trends. Microbiol. 6, 131–1333. 6. Udhayakumar, V., and Muthukkaruppan, V. R. (1987) Protective immunity induced by outer membrane proteins of Salmonella typhimurium in mice. Infect. Immun. 55, 1583–1587. 7. Isibasi, A., Oritz, V., Vargas, M., Paniagua, J., Gonzalez, C., Moreno, J., and Kumate, J. (1988) Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins from Salmonella typhi 9, 12, d, Vi. Infect. Immun. 56, 2953–2959. 8. Puente, J. L., Alvarez-Scherer, V., Gosset, G., and Calva, E. (1989) Comparative analysis of the Salmonella typhi and the Escherichia coli ompC genes. Gene 83, 197–206. 9. Puente, J. L., Juarez, D., Bobadilla, M., Arias, C. F., and Calva, E. (1995) The Salmonella typhi OmpC gene: Structure and uses as a carrier for heterologus sequences. Gene 156, 1–9. 10. Muthukkaruppan, V. R., Nandakumar, K. S., and Palanivel, V. (1992) Monoclonal antibodies against Salmonella porins: Generation and characterization. Immunol. Lett. 33, 201–206. 11. Arockiasamy, A., and Krishnaswamy, S. (1995) Prediction of B-cell epitopes for Salmonella typhi OmpC. J. Biosci. 20, 235– 243. 12. Arockiasamy, A., and Krishnaswamy, S. (1999) Crystallization of the immunodominant outer membrane protein OmpC; the first protein crystals from Salmonella typhi, a human pathogen. FEBS. Lett. 453, 380 –382. 13. Tokunaga, H., Tokunaga, M., and Nakae, T. (1979) Characterization of porins from the outer membrane of Salmonella typhimurium. Eur. J. Biochem. 95, 433– 439. 14. Garavito, R. M., and Rosenbusch, J. P. (1986) Isolation and crystallization of bacterial porin. Methods Enzymol. 125, 309 – 328. 15. Yoshimura, F., Zalman, L. S., and Nikaido, H. (1983) Purification and properties of Pseudomonas aeruginosa Porin. J. Biol. Chem. 258, 2308 –2314. 16. Schiltz, E., Kreusch, A., Nestel, U., and Schulz, G. E. (1991) Primary structure of porin from Rhodobacter capsulatus. Eur. J. Biochem. 999, 587–594. 17. Gelder, P. A., Steiert, M., Khattabi, M. E., Rosenbusch, J. P., and Tommassen, J. (1996) Structural and functional characterization of a His-tagged PhoE pore protein of Escherichia coli. Biochem. Biophys. Res. Commun. 229, 869 – 875. 18. Germanier, R., and Furer, E. (1975) Isolation and Characterization of Gal E mutant Ty21a of Salmonella typhi: A candidate strain for a live, oral typhoid Vaccine. J. Infect. Dis. 131, 553– 558. 19. Lakshmi, Mundkur (1995) Immunological importance of Salmonella porins, Ph.D thesis, Madurai Kamaraj University, Madurai, India. 20. Peterson, G. L. (1983) Determination of total protein. Methods Enzymol. 91, 95–119. 21. Laemmli, G. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 277, 680 – 685. 22. Nandakumar, K. S. (1994) Studies on the importance of porin, an outer membrane protein, in murine and human typhoid, Ph.D thesis, Madurai Kamaraj University, Madurai, India. 23. Butz, S., Benz, R., Wacker, T., Welte, W., Lustig, A., Plapp, R., and Weckesser, J. (1993) Biochemical characterization and crystallization of porin from Rhodopseudomonas blatica. Arch. Microbiol. 159, 301–307. 24. Rosenbusch, J. P. (1974) Characterization of the Major Envelope protein from Escherichia coli. J. Biol. Chem. 24, 8019 – 8029.

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25. Diedrich, A. L., Stein, M. A., and Schnaitman, C. A. (1990) Association of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides. J. Bacteriol. 172, 5307–5311. 26. Lugtenberg, B., and Alphen, L. V. (1983) Molecular architecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria. Biochim. Biophys. Acta 737, 51–115. 27. Lobos, S. R., and Mora, G. C. (1991) Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulphate concentration in sodium dodecyl sulfate poly acrylamide gel electrophoresis. Electrophoresis 12, 448 – 450. 28. Hancock, R. E. W., and Carey, A. M. (1979) Outer membrane of Pseudomonas aeruginosa: Heat and 2-mercapto ethanol-modifiable proteins. J. Bacteriol. 140, 902–910. 29. Arockiasamy, A., Umesh Chandra Reddy, K., and Krishnaswamy, S. (1998) Probing the structural organisation of S.

typhi OmpC using monoclonal antibodies, SDS/urea gel electrophoresis and circular dichroism. Abstracts of XXIX National Seminar on Crystallography, December 1998, University of Madras, Chennai, India, E2.2. 30. Garavito, M., and Rosenbusch, J. P. (1986) Isolation and crystallization of bacterial porin. Methods Enzymol. 125, 309 –328. 31. Rocque, W. J., Coughlin, R. T., and McGroarty, E. J. (1987) Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12. J. Biol. Chem. 169, 4003– 4010. 32. Arockiasamy, A., Murthy, G. S., and Krishnaswamy, S. (1998) Conformational epitope mapping and kinetics of MAb-porin interactions for S. typhi OmpC. Proceedings of the 10th International Congress of Immunology, Nov 1998, New Delhi, India. The Immunologist, Suppl. 1, 20.