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A simplified method for isolating outer membrane proteins from Pasteurella haemolytica A1
Journal of Microbiological Methods 29 (1997) 201–206
Journal of Microbiological Methods
A simplified method for isolating outer membrane proteins from Pasteurella haemolytica A1 a, a b Robert E. Brennan *, Richard E. Corstvet , Jere W. McBride a
Department of Veterinary Science, 111 Dalrymple Building, South Campus Dr., Louisiana State University, Agricultural Center, Baton Rouge, LA 70803, USA b Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Received 5 February 1997; received in revised form 27 May 1997; accepted 27 May 1997
Abstract The outer membrane of Pasteurella haemolytica A1 (Ph-1) contains several major proteins which may play an important role in stimulating protective immunity. This report describes a method which was used to successfully isolate three Ph-1 outer membrane proteins (OMPs). The molecular weights of these OMPs are 31 kilodaltons (kD), 40 kD and 42 kD respectively. This method involves two steps: first, a sarcosyl extraction step was used to obtain a crude OMP prep; and secondly, these OMPs were separated by gel electrophoresis using a Bio-Rad 491 Prep Cell which separates proteins based on their molecular weights. Isolation of the proteins was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogenicity was then demonstrated by immunoblotting and enzyme-linked immunosorbent assays (ELISAs) with convalescent serum taken from calves with prior exposure to Ph-1. SDS-PAGE results show that single bands of each protein were obtained. Immunoblots and ELISAs of these proteins show that the convalescent antiserum used reacted with the 31 kD and 40 kD proteins, but did not react with the 42 kD protein. This procedure provides for a simplified method to obtain highly purified OMPs from Ph-1 which may now be evaluated on an individual basis as to what role they may play in stimulating resistance to pasteurellosis. 1997 Elsevier Science B.V. Keywords: Pasteurella haemolytica A1; Outer membrane proteins; Isolation; ELISA
1. Introduction Pneumonic pasteurellosis has been recognized as a source of major economic loss to the cattle industry since the early 1900’s [1–3]. It is now thought that the primary agent responsible for producing the fibrinous pneumonia associated with this disease is Pasteurella haemolytica A1 (Ph-1) [3]. Attempts to
*Corresponding author. Tel.: 11 504 3884769; fax: 11 504 3884890.
reduce the prevalence of this disease using live vaccines, recombinant vaccines, bacterins, supernatant preparations and subunit preparations have had limited beneficial effects [2]. The inconsistency of these vaccines can in part be attributed to the variability of their antigenic content and lack of knowledge about which antigens are important for stimulating protective immunity [4,5]. Efforts over the last ten to fifteen years have been focused on identifying and characterizing Ph-1 components which may be important for stimulating protective immune responses [4]. Among these components are
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0167-7012( 97 )00050-X
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several outer membrane proteins (OMPs). In many bacteria OMPs are known to play an important role in the infectious process through their ability to act as porins and through their involvement in adhesion of bacteria to host cells [6]. It is thought that antibodies to these OMPs could serve as effective immunogens by preventing adherence, promoting phagocytosis, and causing death of the bacteria by interfering with membrane function [6]. Several studies have indicated that antibodies to Ph-1 OMPs do play a vital role in resistance to pasteurellosis [5,7,8]. In order to identify which proteins are responsible for resistance it is necessary to separate and characterize them [9]. Isolation of these proteins would also be useful in vaccine production as well as for evaluating systemic as well as local antibody responses [10]. Extraction of Ph-1 OMPs was previously reported by Squire et al. [6]. The OMP fraction produced contained two major proteins; one at 30 kD and one at 42 kD. Another study [11] reported that a saline extracted antigen prep contained a 29 kD and a 44 kD protein that were recognized by Ph-1 antiserum by immunoblot. These studies demonstrated that OMPs of Ph-1 are extractable and are immunogenic. The goal of this project was to use a simple method to obtain highly purified proteins from Ph-1 OMP extracts and achieve antigenic yields large enough so that these proteins can be studied on an individual basis with respect to their immunogenicity.
2. Materials and methods
2.1. Organism and growth conditions A lyophilized preparation of Pasteurella haemolytica A1 (Ph-1), originally isolated from an infected calf [12], was suspended in phosphate buffered saline (PBS) pH 7.2 and cultivated on brain heart infusion agar with 5% bovine blood for 18–24 h at 378C. One isolated colony was picked and inoculated into 1 l of BHI broth and cultivated for an additional 18 h at 378C with orbital shaking. Bacteria were then harvested by centrifugation in 50 ml centrifuge tubes at 6000 g for 20 min.
2.2. OMP extraction OMP extraction was performed according to a method previously described [13]. Briefly, sedimented bacteria were pooled and suspended in 35 ml of 20 mM Tris (pH 7.2) and pelleted at 6000 g for 20 min. Bacteria were then resuspended in 35 ml of 20 mM Tris (pH 7.2) and placed in a 50 ml beaker and sonicated with 0.750 probe (55 mm, amplitude setting 9.20 Hz) on ice for 6 min continuously. Sonicated prep was centrifuged at 6000 g for 20 min. The supernate, which contained the cell wall and OMPs was removed with a pipette and placed into a clear ultracentrifuge tube and pelleted at 60 000 g for 1 h. The supernate was then removed and the pellet was resuspended in 1 ml of 20 mM Tris (pH 7.2). Cytoplasmic membranes were solubilized by adding 4 ml of 0.5% N-lauryl-sarcosine and incubating at room temperature for 30 min. Clumps were broken up by pipetting up and down several times. The OMPs were then transferred to a 5 ml ultracentrifuge tube and pelleted at 60 000 g for 1 h and washed once in 20 mM Tris (pH 7.2). The pellet was then resuspended in 1 ml of 20 mM Tris (pH 7.2) and the protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Life Science, Hercules, CA, USA).
2.3. Isolation of OMPs Isolation of the individual proteins from this crude prep was then accomplished using the 491 Prep Cell. The 491 Prep Cell has previously been used by Hager et al. and Yamamoto and Munn to successfully isolate proteins from other agents. [14,15]. First, analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the acrylamide concentration (%T) that would best separate the 31 kD and the 42 kD proteins from the nearest contaminants. It was determined that an 11% resolving gel would give us the best separation. An 11% polyacrylamide resolving gel was cast into a 27 mm gel tube to 10 ml and overlaid with 1 ml of water saturated 2-butanol. This was allowed to polymerize for 3.5 h using a distilled water (dH 2 O) circulation pump as a coolant. The butanol was then decanted and the gel was rinsed
R.E. Brennan et al. / Journal of Microbiological Methods 29 (1997) 201 – 206
two times with dH 2 O. A 4% polyacrylamide stacking gel was cast onto the gel to 14 ml and allowed to polymerize for 3 h cooled by the circulation pump. The 1 ml of crude extract containing approximately 13 mg of protein per ml was diluted to 7 ml in SDS reducing buffer and loaded directly onto the gel using a 20 ml syringe and a 20 14 gauge needle. The sample was electrophoresed for 5 h at 10 W constant power. Seventy 5 ml fractions were collected over a 6 h time period using a 211 Multitrac fraction collector (LKB Bromma, Sweden).
2.4. Identification and immunoblotting of proteins Identification of the individual proteins was achieved by SDS-PAGE. Briefly, 12% polyacrylamide gels 0.75 mm thick were cast using a Mini-Protean II electrophoresis system (Bio-Rad Life Science). A 10 ml volume of individual fractions were loaded per lane and electrophoresed for 45 min at 200 volts. The gels were then stained with Coomassie brilliant blue R-250 (Ameresco, Solon, OH, USA) to visualize proteins. Samples containing the proteins of interest were concentrated into 3 ml samples using Centriprep 3 concentrators (Amicon, Beverly, MA, USA). Immunoblotting was done by performing SDSPAGE on isolated proteins as before and then electrophoretically transferring the proteins to a nitrocellulose membrane (Optitran, Schleicher and Schuell, Keene, NH, USA) using a Bio-Rad TansBlot SD Semi-Dry Transfer Cell. The transfer was run at 18 volts for 30 min. Proteins were reacted with high titer convalescent bovine anti Ph-1 serum and probed with peroxidase labeled goat anti-bovine immunoglobulin G (IgG) (heavy and light) (Kirkegaard and Perry, Gaithersburg, MD, USA) diluted 1:750 in PBS. The substrate 4-chloro-1-napthol (4CN) (Kirkegaard and Perry) was used to reveal protein–antibody reactions.
2.4.1. Enzyme-linked immunosorbent assay ( ELISA) Isolated proteins were diluted to 4 mg / ml in 0.1 M carbonate buffer (pH 9.2) and added at 100 ml / well to 96 well microtitration plates (Dynatech Laboratories, Chantilly, VA, USA). Plates were sealed in a
203
ziplock bag and placed at 48C for 18–24 h for fixation. Plates were then washed three times with dH 2 O1NaCl1tween 20 and blocked with NET (0.1 M NaCl11 mM EDTA110 mM Tris base pH 8.0)1 10% goat serum for 30 min at 378C. After blocking, plates were washed as before and 100 ml of (1) convalescent bovine anti-Ph-1 sera or (2) pre-Ph-1 exposure sera was added to appropriate wells and incubated for 1 h at 378C and then the wells were washed again. The wells were then probed with 100 ml of peroxidase labeled goat anti-bovine IgG (heavy and light) (Kirkegaard and Perry) diluted 1:750 in NET110% goat serum and incubated for 1 h at 378C. After a final washing, 100 ml of ABTS [229azino-di(3 ethylbenzothiazoline sulfone-6)] substrate (Kirkegaard and Perry) was added per well and allowed to develop for 10 min. Optical densities (ODs) were determined using an ELISA reader (MR 5000 microplate reader, Dynatech) at a wavelength of 405 nm.
3. Results
3.1. Purification of proteins After the sarcosyl extraction step previously described we obtained a 1 ml sample which contained approximately 13 mg of crude OMP. Fig. 1 illustrates
Fig. 1. Analysis by SDS-PAGE and Coomassie brilliant blue R-250 staining of the outer membrane prep after sarcosyl extraction step. Lane 1, Ph-1 whole cell organism (20 mg of protein); Lane 2, Ph-1 outer membrane proteins (20 mg of protein).
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the contrast between whole cell Ph-1 and what proteins remain after sarcosyl extraction of the outer membrane. The two major proteins of interest are present in this sample as well as other minor proteins. We were able to separate the 31 kD and 42 kD proteins from the others by preparative gel electrophoresis using an 11% SDS-PAGE in a 491 Prep Cell (Bio-Rad). We were able to obtain two distinct and highly pure samples as seen in Fig. 2. The 31 kD protein was present in fraction numbers 14, 15, and 16, while the 42 kD protein was present in fraction numbers 34, 35, and 36. We obtained a final volume of 3 ml for each sample with protein concentrations of 300 mg / ml for the 31 kD protein and 500 mg / ml for the 42 kD protein. In the process of purifying these two major proteins a 40 kD minor protein was also purified as shown in Fig. 2. This protein was present in fraction numbers 30, 31, and 32. A concentration of 420 mg / ml of this protein was recovered.
3.2. Immunoblotting of proteins The anti-Ph-1 convalescent serum used to probe these purified proteins reacted with the 31 kD and 40
Fig. 3. Immunoblot analysis of isolated proteins after SDS-PAGE and transfer to nitrocellulose membrane. Samples were probed with polyvalent antibody to Ph-1. Lane 1, Ph-1 whole cell; Lane 2, 31 kD protein; Lane 3, 40 kD protein; Lane 4, 42 kD protein.
kD proteins but not the 42 kD protein as shown in Fig. 3.
3.3. ELISA Antibody responses obtained by ELISA from the convalescent (1) and the pre-exposure (2) serum to the 31 kD and 40 kD proteins are expressed in ODs. The ODs of the (1) serum to these two proteins were significantly greater ( p,0.05) than the (2) serum. The (1) serum had ODs of 1.497 and 1.538 respectively while the (2) serum ODs were 0.476 and 0.623. Results are illustrated in Fig. 4.
4. Discussion
Fig. 2. Analysis by SDS-PAGE and Coomassie brilliant blue R-250 staining of the outer membrane proteins after isolation with Bio-Rads 491 Prep Cell. Lane 1, molecular weight marker (BioRad); Lane 2, Ph-1 whole cell (5 g of protein); Lane 3, 31 kD protein (3 mg of protein); Lane 4, 40 kD protein (4 mg of protein); Lane 5, 42 kD protein (5 mg of protein).
This report has described a two-step procedure for the purification of Ph-1 OMPs which provides us with the ability to obtain highly purified and immunologically stable proteins. Advantages that we feel this technique has over other techniques that have been used to extract proteins from Ph-1 such as
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Fig. 4. Comparison between (2) serum ODs and (1) serum ODs to the 31 kD and 40 kD isolated proteins. Results obtained by ELISA.
sucrose density gradients, isoelectrofocusing, and chromatofocusing [6,11] are the ability to obtain purer proteins, achieve higher antigenic yields, and be less laborious. The proteins purified in this manner were recovered in quantities that will allow for use in biological assays (i.e. ELISAs and Western blots). Also, there are several other studies which have proposed that OMPs from a number of organisms may be suitable components for effective vaccines [16–18], which may make this technique even more desirable. One drawback that we experienced during this study was the amount of time it took to identify which fractions contained the proteins of interest which can be attributed to our inability to monitor the proteins as they were eluted from the 491 Prep Cell. This resulted in the screening of all of the fractions until we identified which ones contained the relevant proteins. The incorporation of a UV monitor and a chart recorder would have allowed us to determine which fractions contained protein and which ones did not, ultimately reducing the number of fractions needing to be screened. As mentioned before it will now be possible to begin to investigate, on an individual basis, the involvement of these OMPs as well as other Ph-1 proteins in the pathogenesis of and the resistance to Ph-1. Currently two of the OMPs that we have isolated (31 kD and 40 kD) are being incorporated into ELISA and Western blot assays in our lab. This does not suggest that the 42 kD protein is not important; only that it requires further investigation.
The lack of antibody response to this protein as measured by immunoblot and ELISA may be due to the fact that the immune system in the animal from which we collected the convalescent sera did not recognize this protein. It may also be that this protein is not an immunologically dominant antigen or that it may be part of a highly conformational protein which when denatured during SDS-PAGE is no longer immunologically active. In conclusion, this study has described a simplified method with which we were able to isolate OMPs from Pasteurella haemolytica A1. The 31 kD and 40 kD proteins are being used to screen samples (nasal, lavage, serum, and tonsil) from calves that have been experimentally exposed to Ph-1 and to look at the local antibody responses to these proteins and how they may correlate to resistance from pasteurellosis. These studies could provide valuable information in the effort to develop new and improved vaccines in which the identification of which components of Ph-1 stimulate protective immunity appears to be crucial.
References [1] A.W. Confer, R.J. Panciera, Testing of two new generation Pasteurella haemolytica vaccines against experimental bovine pneumonic pasteurellosis, Agri. Pract. Immunol. 15(8) (1994) 10–15. [2] D.A. Mosier, A.W. Confer, R.W. Panciera, The evolution of vaccines for bovine pneumonic pasteurellosis, Res. Vet. Sci. 47 (1989) 1–10.
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[3] L.O. Whitely, S.K. Maheswaran, D.J. Weiss, T.R. Ames, M.S. Kannan, Pasteurella haemolytica A1 and bovine respiratory disease: pathogenesis, J. Vet. Int. Med. 6 (1992) 11–22. [4] D.A. Mosier, K.R. Simons, A.W. Confer, R.J. Panciera, K.D. Clinkenbeard, Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis. Infect. Immunity Mar. 57 (1989) 711–716. [5] D.A. Mosier, K.R. Simons, M.M. Chengappa, A.W. Confer, Antigenic composition of Pasteurella haemolytica serotype1 supernatants from supplemented and nonsupplemented media, Am. J. Vet. Res. 55(3) (1994) 348–352. [6] P.G. Squire, D.W. Smiley, R.B. Croskell, Identification and extraction of Pasteurella haemolytica membrane proteins, Infect. Immunity Sept. (1984) 667–673. [7] Y.S. Lu, H.N. Aguila, W.C. Lai, S.P. Pakes, Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice, Infect. Immunity Apr. (1991) 1470–1475. [8] K.R. Simons, R.J. Morton, R.W. Fulton, A.W. Confer, Comparison of antibody responses in cattle to outer membrane proteins from Pasteurella haemolytica serotype-1 and from eight untypeable strains, Am. J. Vet. Res. 53 (1992) 971–975. [9] A.W. Confer, B.A. Lesley, R.W. Panciera, J.A. Fulton, J.A. Kreps, Serum antibodies to antigens derived from a saline extract of Pasteurella haemolytica: Correlation with resistance to experimental bovine pneumonic pasteurellosis, Vet. Immunol. Immunopathol. 10 (1985) 265–278. [10] K.L. McKinney, A.W. Confer, J.A. Rummage, M.J. Gentry, J.A. Durham, Pasteurella haemolytica: Purification of salineextractable proteins by isoelectrofocusing, Vet. Microbiol. 10 (1985) 465–480. [11] B.A. Lessley, A.W. Confer, D.A. Mosier, M.J. Gentry, J.A.
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Durham, J.A. Rummage, Saline-extracted antigens of Pasteurella haemolytica: Separation by chromatofocusing, preliminary characterization and evaluation of immunogenicity, Vet. Immunol. Immunopathol. 10 (1985) 279–296. P.R. Newman, R.E. Corstvet, R.J. Panciera, Distribution of Pasteurella haemolytica and Pasteurella multocida in the bovine lung following vaccination and challenge exposure as an indicator of lung resistance, Am. J. Vet. Res. 43 (1982) 417–422. R.L. Davies, Outer membrane protein profiles of Yersinia ruckeri, Vet. Microbiol. 26 125–146. K.M. Hagar, E.M. Wright, Preparative SDS gel electrophoresis of sodium / glucose cotransporter fusion protein. Bio-Rad Preparative Electrophoresis Review. US / EG Bulletin, 1995, p. 1685. J. Yamamoto, R.J. Munn, Isolation of a FAIDS upregulated protein from infected feline lymphoid cell lysates by preparative SDS gel electrophoresis. Bio-Rad Preparative Electrophoresis Review. US / EG Bulletin, 1995, p. 1685. J.M. Kyd, D. Taylor, A.W. Cripps, Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae, Infect. Immunity 62 (1994) 5652– 5658. R. Anderson, G. Dougan, M. Roberts, Delivery of the pertactin / P.69 polypeptide of Bordetella pertussis using an attenuated Salmonella typhimurium vaccine strain: Expression levels and immune response, Vaccine 14 (1996) 1384– 1390. Y.Q. Zhang, D. Mathieson, C.P. Lolbert, J. Anderson, R.T. Shoen, E. Kikrig, D.H. Persing, Borrelia burgdorferi enzyme-linked immunosorb and assay for discrimination of OspA vaccination from spirochete infection, J. Clin. Microbiol. 1 (1997) 233–238.
Journal of Microbiological Methods
A simplified method for isolating outer membrane proteins from Pasteurella haemolytica A1 a, a b Robert E. Brennan *, Richard E. Corstvet , Jere W. McBride a
Department of Veterinary Science, 111 Dalrymple Building, South Campus Dr., Louisiana State University, Agricultural Center, Baton Rouge, LA 70803, USA b Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Received 5 February 1997; received in revised form 27 May 1997; accepted 27 May 1997
Abstract The outer membrane of Pasteurella haemolytica A1 (Ph-1) contains several major proteins which may play an important role in stimulating protective immunity. This report describes a method which was used to successfully isolate three Ph-1 outer membrane proteins (OMPs). The molecular weights of these OMPs are 31 kilodaltons (kD), 40 kD and 42 kD respectively. This method involves two steps: first, a sarcosyl extraction step was used to obtain a crude OMP prep; and secondly, these OMPs were separated by gel electrophoresis using a Bio-Rad 491 Prep Cell which separates proteins based on their molecular weights. Isolation of the proteins was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogenicity was then demonstrated by immunoblotting and enzyme-linked immunosorbent assays (ELISAs) with convalescent serum taken from calves with prior exposure to Ph-1. SDS-PAGE results show that single bands of each protein were obtained. Immunoblots and ELISAs of these proteins show that the convalescent antiserum used reacted with the 31 kD and 40 kD proteins, but did not react with the 42 kD protein. This procedure provides for a simplified method to obtain highly purified OMPs from Ph-1 which may now be evaluated on an individual basis as to what role they may play in stimulating resistance to pasteurellosis. 1997 Elsevier Science B.V. Keywords: Pasteurella haemolytica A1; Outer membrane proteins; Isolation; ELISA
1. Introduction Pneumonic pasteurellosis has been recognized as a source of major economic loss to the cattle industry since the early 1900’s [1–3]. It is now thought that the primary agent responsible for producing the fibrinous pneumonia associated with this disease is Pasteurella haemolytica A1 (Ph-1) [3]. Attempts to
*Corresponding author. Tel.: 11 504 3884769; fax: 11 504 3884890.
reduce the prevalence of this disease using live vaccines, recombinant vaccines, bacterins, supernatant preparations and subunit preparations have had limited beneficial effects [2]. The inconsistency of these vaccines can in part be attributed to the variability of their antigenic content and lack of knowledge about which antigens are important for stimulating protective immunity [4,5]. Efforts over the last ten to fifteen years have been focused on identifying and characterizing Ph-1 components which may be important for stimulating protective immune responses [4]. Among these components are
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0167-7012( 97 )00050-X
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several outer membrane proteins (OMPs). In many bacteria OMPs are known to play an important role in the infectious process through their ability to act as porins and through their involvement in adhesion of bacteria to host cells [6]. It is thought that antibodies to these OMPs could serve as effective immunogens by preventing adherence, promoting phagocytosis, and causing death of the bacteria by interfering with membrane function [6]. Several studies have indicated that antibodies to Ph-1 OMPs do play a vital role in resistance to pasteurellosis [5,7,8]. In order to identify which proteins are responsible for resistance it is necessary to separate and characterize them [9]. Isolation of these proteins would also be useful in vaccine production as well as for evaluating systemic as well as local antibody responses [10]. Extraction of Ph-1 OMPs was previously reported by Squire et al. [6]. The OMP fraction produced contained two major proteins; one at 30 kD and one at 42 kD. Another study [11] reported that a saline extracted antigen prep contained a 29 kD and a 44 kD protein that were recognized by Ph-1 antiserum by immunoblot. These studies demonstrated that OMPs of Ph-1 are extractable and are immunogenic. The goal of this project was to use a simple method to obtain highly purified proteins from Ph-1 OMP extracts and achieve antigenic yields large enough so that these proteins can be studied on an individual basis with respect to their immunogenicity.
2. Materials and methods
2.1. Organism and growth conditions A lyophilized preparation of Pasteurella haemolytica A1 (Ph-1), originally isolated from an infected calf [12], was suspended in phosphate buffered saline (PBS) pH 7.2 and cultivated on brain heart infusion agar with 5% bovine blood for 18–24 h at 378C. One isolated colony was picked and inoculated into 1 l of BHI broth and cultivated for an additional 18 h at 378C with orbital shaking. Bacteria were then harvested by centrifugation in 50 ml centrifuge tubes at 6000 g for 20 min.
2.2. OMP extraction OMP extraction was performed according to a method previously described [13]. Briefly, sedimented bacteria were pooled and suspended in 35 ml of 20 mM Tris (pH 7.2) and pelleted at 6000 g for 20 min. Bacteria were then resuspended in 35 ml of 20 mM Tris (pH 7.2) and placed in a 50 ml beaker and sonicated with 0.750 probe (55 mm, amplitude setting 9.20 Hz) on ice for 6 min continuously. Sonicated prep was centrifuged at 6000 g for 20 min. The supernate, which contained the cell wall and OMPs was removed with a pipette and placed into a clear ultracentrifuge tube and pelleted at 60 000 g for 1 h. The supernate was then removed and the pellet was resuspended in 1 ml of 20 mM Tris (pH 7.2). Cytoplasmic membranes were solubilized by adding 4 ml of 0.5% N-lauryl-sarcosine and incubating at room temperature for 30 min. Clumps were broken up by pipetting up and down several times. The OMPs were then transferred to a 5 ml ultracentrifuge tube and pelleted at 60 000 g for 1 h and washed once in 20 mM Tris (pH 7.2). The pellet was then resuspended in 1 ml of 20 mM Tris (pH 7.2) and the protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Life Science, Hercules, CA, USA).
2.3. Isolation of OMPs Isolation of the individual proteins from this crude prep was then accomplished using the 491 Prep Cell. The 491 Prep Cell has previously been used by Hager et al. and Yamamoto and Munn to successfully isolate proteins from other agents. [14,15]. First, analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the acrylamide concentration (%T) that would best separate the 31 kD and the 42 kD proteins from the nearest contaminants. It was determined that an 11% resolving gel would give us the best separation. An 11% polyacrylamide resolving gel was cast into a 27 mm gel tube to 10 ml and overlaid with 1 ml of water saturated 2-butanol. This was allowed to polymerize for 3.5 h using a distilled water (dH 2 O) circulation pump as a coolant. The butanol was then decanted and the gel was rinsed
R.E. Brennan et al. / Journal of Microbiological Methods 29 (1997) 201 – 206
two times with dH 2 O. A 4% polyacrylamide stacking gel was cast onto the gel to 14 ml and allowed to polymerize for 3 h cooled by the circulation pump. The 1 ml of crude extract containing approximately 13 mg of protein per ml was diluted to 7 ml in SDS reducing buffer and loaded directly onto the gel using a 20 ml syringe and a 20 14 gauge needle. The sample was electrophoresed for 5 h at 10 W constant power. Seventy 5 ml fractions were collected over a 6 h time period using a 211 Multitrac fraction collector (LKB Bromma, Sweden).
2.4. Identification and immunoblotting of proteins Identification of the individual proteins was achieved by SDS-PAGE. Briefly, 12% polyacrylamide gels 0.75 mm thick were cast using a Mini-Protean II electrophoresis system (Bio-Rad Life Science). A 10 ml volume of individual fractions were loaded per lane and electrophoresed for 45 min at 200 volts. The gels were then stained with Coomassie brilliant blue R-250 (Ameresco, Solon, OH, USA) to visualize proteins. Samples containing the proteins of interest were concentrated into 3 ml samples using Centriprep 3 concentrators (Amicon, Beverly, MA, USA). Immunoblotting was done by performing SDSPAGE on isolated proteins as before and then electrophoretically transferring the proteins to a nitrocellulose membrane (Optitran, Schleicher and Schuell, Keene, NH, USA) using a Bio-Rad TansBlot SD Semi-Dry Transfer Cell. The transfer was run at 18 volts for 30 min. Proteins were reacted with high titer convalescent bovine anti Ph-1 serum and probed with peroxidase labeled goat anti-bovine immunoglobulin G (IgG) (heavy and light) (Kirkegaard and Perry, Gaithersburg, MD, USA) diluted 1:750 in PBS. The substrate 4-chloro-1-napthol (4CN) (Kirkegaard and Perry) was used to reveal protein–antibody reactions.
2.4.1. Enzyme-linked immunosorbent assay ( ELISA) Isolated proteins were diluted to 4 mg / ml in 0.1 M carbonate buffer (pH 9.2) and added at 100 ml / well to 96 well microtitration plates (Dynatech Laboratories, Chantilly, VA, USA). Plates were sealed in a
203
ziplock bag and placed at 48C for 18–24 h for fixation. Plates were then washed three times with dH 2 O1NaCl1tween 20 and blocked with NET (0.1 M NaCl11 mM EDTA110 mM Tris base pH 8.0)1 10% goat serum for 30 min at 378C. After blocking, plates were washed as before and 100 ml of (1) convalescent bovine anti-Ph-1 sera or (2) pre-Ph-1 exposure sera was added to appropriate wells and incubated for 1 h at 378C and then the wells were washed again. The wells were then probed with 100 ml of peroxidase labeled goat anti-bovine IgG (heavy and light) (Kirkegaard and Perry) diluted 1:750 in NET110% goat serum and incubated for 1 h at 378C. After a final washing, 100 ml of ABTS [229azino-di(3 ethylbenzothiazoline sulfone-6)] substrate (Kirkegaard and Perry) was added per well and allowed to develop for 10 min. Optical densities (ODs) were determined using an ELISA reader (MR 5000 microplate reader, Dynatech) at a wavelength of 405 nm.
3. Results
3.1. Purification of proteins After the sarcosyl extraction step previously described we obtained a 1 ml sample which contained approximately 13 mg of crude OMP. Fig. 1 illustrates
Fig. 1. Analysis by SDS-PAGE and Coomassie brilliant blue R-250 staining of the outer membrane prep after sarcosyl extraction step. Lane 1, Ph-1 whole cell organism (20 mg of protein); Lane 2, Ph-1 outer membrane proteins (20 mg of protein).
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the contrast between whole cell Ph-1 and what proteins remain after sarcosyl extraction of the outer membrane. The two major proteins of interest are present in this sample as well as other minor proteins. We were able to separate the 31 kD and 42 kD proteins from the others by preparative gel electrophoresis using an 11% SDS-PAGE in a 491 Prep Cell (Bio-Rad). We were able to obtain two distinct and highly pure samples as seen in Fig. 2. The 31 kD protein was present in fraction numbers 14, 15, and 16, while the 42 kD protein was present in fraction numbers 34, 35, and 36. We obtained a final volume of 3 ml for each sample with protein concentrations of 300 mg / ml for the 31 kD protein and 500 mg / ml for the 42 kD protein. In the process of purifying these two major proteins a 40 kD minor protein was also purified as shown in Fig. 2. This protein was present in fraction numbers 30, 31, and 32. A concentration of 420 mg / ml of this protein was recovered.
3.2. Immunoblotting of proteins The anti-Ph-1 convalescent serum used to probe these purified proteins reacted with the 31 kD and 40
Fig. 3. Immunoblot analysis of isolated proteins after SDS-PAGE and transfer to nitrocellulose membrane. Samples were probed with polyvalent antibody to Ph-1. Lane 1, Ph-1 whole cell; Lane 2, 31 kD protein; Lane 3, 40 kD protein; Lane 4, 42 kD protein.
kD proteins but not the 42 kD protein as shown in Fig. 3.
3.3. ELISA Antibody responses obtained by ELISA from the convalescent (1) and the pre-exposure (2) serum to the 31 kD and 40 kD proteins are expressed in ODs. The ODs of the (1) serum to these two proteins were significantly greater ( p,0.05) than the (2) serum. The (1) serum had ODs of 1.497 and 1.538 respectively while the (2) serum ODs were 0.476 and 0.623. Results are illustrated in Fig. 4.
4. Discussion
Fig. 2. Analysis by SDS-PAGE and Coomassie brilliant blue R-250 staining of the outer membrane proteins after isolation with Bio-Rads 491 Prep Cell. Lane 1, molecular weight marker (BioRad); Lane 2, Ph-1 whole cell (5 g of protein); Lane 3, 31 kD protein (3 mg of protein); Lane 4, 40 kD protein (4 mg of protein); Lane 5, 42 kD protein (5 mg of protein).
This report has described a two-step procedure for the purification of Ph-1 OMPs which provides us with the ability to obtain highly purified and immunologically stable proteins. Advantages that we feel this technique has over other techniques that have been used to extract proteins from Ph-1 such as
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Fig. 4. Comparison between (2) serum ODs and (1) serum ODs to the 31 kD and 40 kD isolated proteins. Results obtained by ELISA.
sucrose density gradients, isoelectrofocusing, and chromatofocusing [6,11] are the ability to obtain purer proteins, achieve higher antigenic yields, and be less laborious. The proteins purified in this manner were recovered in quantities that will allow for use in biological assays (i.e. ELISAs and Western blots). Also, there are several other studies which have proposed that OMPs from a number of organisms may be suitable components for effective vaccines [16–18], which may make this technique even more desirable. One drawback that we experienced during this study was the amount of time it took to identify which fractions contained the proteins of interest which can be attributed to our inability to monitor the proteins as they were eluted from the 491 Prep Cell. This resulted in the screening of all of the fractions until we identified which ones contained the relevant proteins. The incorporation of a UV monitor and a chart recorder would have allowed us to determine which fractions contained protein and which ones did not, ultimately reducing the number of fractions needing to be screened. As mentioned before it will now be possible to begin to investigate, on an individual basis, the involvement of these OMPs as well as other Ph-1 proteins in the pathogenesis of and the resistance to Ph-1. Currently two of the OMPs that we have isolated (31 kD and 40 kD) are being incorporated into ELISA and Western blot assays in our lab. This does not suggest that the 42 kD protein is not important; only that it requires further investigation.
The lack of antibody response to this protein as measured by immunoblot and ELISA may be due to the fact that the immune system in the animal from which we collected the convalescent sera did not recognize this protein. It may also be that this protein is not an immunologically dominant antigen or that it may be part of a highly conformational protein which when denatured during SDS-PAGE is no longer immunologically active. In conclusion, this study has described a simplified method with which we were able to isolate OMPs from Pasteurella haemolytica A1. The 31 kD and 40 kD proteins are being used to screen samples (nasal, lavage, serum, and tonsil) from calves that have been experimentally exposed to Ph-1 and to look at the local antibody responses to these proteins and how they may correlate to resistance from pasteurellosis. These studies could provide valuable information in the effort to develop new and improved vaccines in which the identification of which components of Ph-1 stimulate protective immunity appears to be crucial.
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