90
Electrophoresis 1991, I2,90-93
M. H. Groschup et al.
1211 Flynn, T. G., Biochem. Pharm. 1982,31,2705-2712. [221 Markus, H. B., Raducha, M. and Harris, H.,Biochem. Med. 1983,29, 31-45. 1231 Sato, S. and Kador, P. F., Invest. Ophthal. Vis. Sci. 1989, 30, 1618- 1622. [241 Tanimoto, T., Fukuda, H. and Kawamura, J., Chem. Pharm. Bull. 1983,31,2395-2403. [251 Schade, S. Z., Early, S. L., Williams, T. R., Kezdy, F. J., Heinrikson, R. L., Grimshaw, C. E. and Doughty, C. C.,J. Biol. Chem. 1990,265, 3628-3635. [261 Carper,D.,Nishimura,C.,Shinohara,T.,Dietzchold,B., Wistow,G., Craft, C., Kador, P. and Kinoshita, J. H., FEBS Lett. 1987, 220, 209-213. [271 Bohren, K. M., Bullock, B., Wermuth, B. and Gabbay, K. H.,J. Bid. Chem. 1989,264,9547-9551.
Martin H. Groschup Jeff Boschwitz John F. Timoney New York State College ofveterinary Medicine, Cornell University, Ithaca, NY
1281 Garcia-Perez, A., Martin, B., Murphy, H. R., Uchida, S., Murer, H.. Cowley, B. D., Handler, J . S. and Burg, M.B., J.Biol. Chem. 1989, 264, 16 815-16 821. [291 Nishimura, C., Graham, C., Hohman, T. C., Nagata, M., Robison, W. G. and Carper, D., Biochem. Biophys. Res. Commun. 1988,153, 1051-1059. [301 Tarle, I., Skow, L. C. and Petrash, J. M., Invest. Ophthal. Vis. Sci. 1990,31, (Suppl.), 353. [311 Kador, P. F. and Sharpless, N. E., Mol. Pharmacol. 1983, 24, 521-53 1. [321 DeLucas, L. J., Bowling, E. and Petrash, J. M., Invest. Ophthal. Vis. Sci. 1987,28, (Suppl.), 87. [331 DeLucas, L. J., Dissertation, University of Alabama at Birmingham, 1981.
A convenient gel holder for preparative electrophoretic separation of aggregated bacterial proteins A simple custom-made gel holder for preparative SDS-PAGE to separate aggregated bacterial antigens is described. The gel holder fits easily into commercially available gel electrophoresis apparatus and proteins or peptides are collected in a stream of distilled water through a channel in the gel. The performance ofthe device was illustrated by the successful separation and purification of fragments of aggregated Streptococprotective antigens. Yields of cus equi M protein and of Erysipelothrix rhusio~ath~ae up to 1.2 mg per run were obtained. The purified proteins retained immunological reactivity and were of sufficient purity for amino acid compositional and sequence analysis.
1 Introduction Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used and effective technique for the separation and resolution ofproteins by their molecular weight. Attempts to scale up this method to the preparative mode have utilized separation on an analytical vertical SDS-PAGE slab gel with subsequent visualization and electroelution of the desired protein band. In commercially available “successive zone elution” analytical scale devices the eluting band is collected in a single step into dialysis tubing at the lower end of the resolving gel. However, the time to harvest, which is critical to the success of the separation, is difficult to control. In addition, the yields using this method are poor. An apparatus for true preparative electrophoresis should allow the application of greater amounts of sample with continuous elution of the separated bands from the resolving gel. A variety of apparatus, based on cylindrical gel columns with cooling systems, have been described [ 1-1 11. Such equipment is highly complex and expensive.
Correspondence: Dr. M. H. Groschup, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 18453, USA Abbreviations: PBS, phosphate buffered saline; PAGE, polyacrylamide gel electrophoresis; SDS,sodium dodecyl sulfate
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Flat gel systems exhibit better heat dissipation due to their greater surface area [12-141. A flat gel modification device described recently [ 15I permits continuous collection of bands from the lower end of the gel following a scaled-up analytical run. This paper describes the successful purification of components of aggregated bacterial antigens of Streptococcus equi and Erysipelothrix rhusiopathiae by using a simple custom-made vertical flat-bed gel holder designed for preparative electrophoresis.
2 Methods 2.1 The preparative gel apparatus
The gel holder (Fig. 1) was designed to fit into a conventional vertical gel electrophoresis apparatus (Hoefer SE 400 “The Sturdier”) and consisted of two acrylic plates (17.6 x 16.1 x 0.45 cm) with acrylic spacers (5.5 x 11 x 0.45 cm and 5.5 x 4.6 x 0.45 cm, respectively) glued between to provide space (9 x 6.6 x 0.45 cm) for the stacking and resolving gel. A transverse horizontal channel for elution of bands during the run was positioned 4.6 cm from the bottom of the gel holder. The gels were poured using the discontinuous buffer system of Laemmli [ 161 and set up as follows. A 24 % acrylamide solution was poured below the channel to prevent the escape of proteins during the elution process. A bar (14.5 x 0.45 x 0.45 0173-0835/9l/010l-0090 %3.50+.25/0
Preparative electrophorev\ of aggregated bacterial proteins
Electrophoresis 199l , I 2 , YO-93
r-
17.6 cm
12.3
91
Electrophoresis and elution procedure
1
pq 16.1
7’
Figurel. Sketchshowing thegeiholder.Spaceforthestacking(l),resolving ( 2 ) and supporting gel (3) is provided between two Plexiglas plates glued together by four spacers. Eluting bands are harvested through a horizontal channel(4) thatisclosedattheinlet(5)andoutletsides(6)bybushings(7,7’) with polyethylene tubing (8,8‘). The gel holder is fixed by screws (9”) into clamps (9) with slots for cams (9”’) that fit into commercially available gel electrophoresis apparatus.Access to the inlet and outlet ports is via openings on the side of the clamps (9’).
The samples in a total volume of approximately 2 mL were applied to the top of the stacking gel (4 % acrylamide) and separated at 35 mA until the dye front entered the collecting channel (0 rnin). The current was then adjusted to 45 mA, the channel was rinsed for 12 rnin with distilled water (9 mL/ min) toflushoutelutedSDS, and proteins andfractions(9mL) were collected at the expected time of elution of the desired band as determined previously by running cytochrome oxidase and bovine serum albumin prestained with Remazol Blue. The approximate elution times of the 66-64 kDa band from alkaline extracts of E. rhusiopathiae was 48-62 min in the 8% polyacrylamide gel with 0.4 % SDS. The 4 I kDa band band from acid extracts of S . equi CF32 eluted after 12-25 min from the 8 % polyacrylamide gel with 0.03 % SDS. All collected fractions were lyophilized (Speedvac Concentrator System, Savant, NY), resolubilized in 100 pL distilled water and analyzed (20 pL) by SDS-PAGE (12 % or 10 %).
2.4 Immunoblotting
Proteins were electrophoretically transferred to nitrocellulose cm) coated with polytetrafluoroethylene was inserted from the [ 191 and immunoblotted. Immunoblots were prepared with port at the side to provide space for the channel between the porcine antiserum to E. rhusiopathiae (WDT, Hoyerhagen, supporting and the resolving gels. The holes at the sides were Germany) followed by peroxidase conjugated protein A (Bioquickly plugged by plastic bushings and the acrylamide solu- Rad, Munich) or rabbit antiserum to S. equi M protein [201 tion was poured to a level of 1 mm above the bar to com- followed by peroxidase conjugated goat anti-rabbit serum pensate for contraction during polymerization. The resolving (Zymed, San Francisco, CA), respectively. Immunoreactive gel of the desired polyacrylamide and SDS concentrations bands were visualized with 4-chloro- 1-naphthol as substrate. was then poured to a height of 6 cm, overlayered with distilled Fractions containing the desired band were pooled and prowater, and allowed to polymerize at room temperature for at tein yields determined by the method of Lowry et al. [21]. least 4 h. Finally, the stacking gel (4 % polyacrylamide) was added. Before removal from the channel, the bar was pushed back and forth several times, using a stick to dislodge scraps of 3 Results and discussion polymerized polyacrylamide. The channel was then flooded with tank buffer (0.1 % SDS in 0.75 M Tris, pH 8.6), air 3.1 Preparative electrophoresis bubbles were driven out and the outlets closed by two plastic bushings with holes drilled through them to accommodate E. rhusiopathiae is an important gram positive pathogen of polyethylene tubing. The inlet side was connected to a distilled pigs and turkeys and carries a protective antigen in the form water reservoir. Fractions of 9 mL were collected into of a glycolipoprotein complex of approximately 200 kDa disposable tubes via a tube from the outlet side. During collec- molecular mass which can separateinto smaller subunits 1221. tion, distilled water or tank buffer diluted 1:100 in distilled Efforts to purify these subunits from extracts by conventional water was flushed through the channel at a rate of 9 ml/min, chromatography resulted in a dramatic decrease of their prothe flow rate being controlled by raising or lowering the reser- tective ability. Our work, using the gel holder and procedure voir attached to the entry port. described above, represents the first successful purification of a protective subunit (66-64 kDa) from alkaline extracts ofE. rhusiopathiae in which protective activity in a mouse protection model was retained (Groschup et al., unpublished data). 2.2 Sample preparation Yields of 0.2-1.2 mg per run were achieved (Fig. 2). An alkaline (0.01 M NaOH) extract 171 of E. rhusiopathiae strain T 28 cells, grown in an overnight (20 h/37 “C) culture Hot acid extraction is a commonly used method to obtain M (500 ml) 1181, was concentrated by ultrafiltration to 1 mL protein from S . equi [201, but it degrades this protein into volume, mixed with an equal volume of double-strength several hydrophobic fragments which tend to aggregate in Laemmli sample buffer [ 161and incubated for 30min at 56 OC aqueous solution. Attempts at purifying individual fragments before application to the gel holder. The SDS and poly- using ion exchange 1231, reverse phase (Timoney et al., unacrylamide concentrations were 0.4 % and 8 %, respectively. published data) and gel sieve exclusion chromatography 120, In the case of S . equi strain CF32, bacterial cells from one L 241 have been unsuccessful due to elution of the fragments as Todd Hewitt broth were extracted by hot acid (HCl, pH 2.5, aggregates in a single peak. Preparative electrophoresis using 95 “C, 10 min). Proteins that adsorbed to hydroxylapatite the custom built gel holder enabled the purification of a 4 1kDa (Bio-Rad, San Francisco, CA) in 10 mM sodium phosphate fragment of S . equiM protein (Fig. 3). This fragment has been buffer, pH 7.2, were eluted with 0.5 M sodium phosphate, pH implicated in protective nasopharyngeal mucosal antibody 9.0, desalted, and loaded in the gel holder containing 8 % responses [241. Quantities of approximately 50- 100 pg purified fragment per run were obtained and were suficient for anpolyacrylamide and 0.03 % SDS resolving gel.
92
Electrophoresis 1991,12, 90-93
M. H. Groschup et al.
Figure 2.Immunoblot offractionsfrom apreparative electrophoresisrun of an alkaline extract of E. rhusiopathiae. One fifth of the harvested fractions (9 mL) were lyophilized and resolubilized in distilled water, separated by SDS-PAGE(I0 %),transferredto nitrocelluloseandimmunoblotted with a porcine antiserum to E. rhusiopathiae (1 :100 in phosphate-buffered saline (PBS) -0.1 Yo Tween 20), followed by protein A (1:1000 in PBS-0.1 % Tween 20) and 4-chloro-1-naphthol as substrate. The numbers on top are the elution times of the fractions in minutes, those on the side are the approximate molecular masses x 1000(kDa). An immunoblot of untreated extract is shown on the right.
Figure3. Immunoblot offractionsfrom apreparativeelectrophoresisrunof a hot acid extract of S. equi. Aliquots of the harvested, lyophilized and resolubilized fractions were separated by SDS-PAGE (10 %), transferred to nitrocellulose and immunoblotted with a rabbit antiserum to S. equi M protein (1 :200 in PBS-0.1 % Tween 20), goat anti-rabbitconjugated peroxidase (1:1000 in PBS-0.1 %Tween 20) and 4-chloro-I-naphthol as substrate. The numbers on top are the elution time of the fractions in minutes, those on the side the approximate molecular weight x 1000(kDa). An immunoblot of nonfractionated extract is shown on track (A).
tiserum production and amino acid compositional and sequence analysis [251.
minimized exposure time to the acrylamide and shortened the fractionation time. Instead of a bar, a transverse elution channel can also be formed by layering the resolving gel on top of a dense sucrose solution during the pouring process. However, an additional step is necessary to thoroughly wash the channel free of sucrose. Either method of forming the channel gave good results, the channel being free of debris and patent during the run. Adhesion of acrylamide to the bar was not a problem at concentrations of acrylamide between 7 and 18 %.The high acrylamide concentration of the supporting gel and the high flow rate (9 mL/min) through the harvesting channel enhanced both separation and yields. SDS is washed off before fractions were collected. If necessary, SDS residues in the harvested band can easily be removed by column chromatography on Extracti-GelTMD (Pierce, Rockford, IL) detergent removing gel. Antigen peaks eluted at highly predictable times during different runs and thus reduced the number of fractions that had to be collected. The recovery rate was 25-40 % when ovalbumin was separated from bovine albumin and cytochrome oxidase. Heat dissipation during runs was adequate due to the favorable volume-to-surface-area ratio of the gel. Additional cooling, if necessary, could easily be provided by attaching a cooling plate to either the freely accessible front or back of the gel holder surface. Because of the relatively narrow width of the gel “smile” effects were negligible. This is an advantage of the custom-made gel holder compared with commercially available vertical gel apparatus fitted with ported spacer kits, where curvature of the bands can result in contamination during elution. The short length of the harvesting channel reduces the time available for the migrating protein to enter the supporting gel during harvesting. This potential source of protein loss is therefore minimized, compared to that which occurs in a wider gel apparatus.
4 Concluding remark In conclusion, preparative electrophoresis with the inexpensive custom-made gel holder provides a simple solution to the separation of aggregated surface components with retention of immunological activity, such as described from E . rhusiopathiae and S . equi.
Supported in part by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to M.H.G.). Thefinancia1 support of the Zweig Memorial Fund is also gratefully acknowledged. Received May 23, 1990
3.2 Construction and operation of the gel holder The gel holder was constructed to fit easily into commercially available gel electrophoresis apparatus such as “The Sturdier”. Leakage from the upper electrode buffer reservoir was prevented by the action of cams inserted in slots in the clamps to compress the rubber gasket between the gel holder and the upper buffer tank. The gel holder could be constructed of glass or, as in the apparatus presented, of Plexiglas. Polycarbonate (lexan) plates were found to be unsuitable because this material blocks the polymerization of polyacrylamide, probably by release of surface-fixed oxygen. Relatively low acrylamide concentrations in the resolving gel were utilized for separating the components of each bacterial extract. This
5 References 111 Jovin, T., Chrambach, A. and Naughton, M. A., Anal. Biochem. 1964,9,351-369. 121 Frattali, V. and Steiner, R. F., Anal. Biochem. 1969,27,285-291. [31 Coy, D.H. and Wuu,T. C., Anal. Biochem. 1971,44,174-181. [41 Vogel, 0.and Reinmuth, H., Anal. Biochem. 1974,62,461-471. [51 Ryan, T.E., Woods, G. M., Kirkpatrick, F. H. and Scamoo, A. E., Anal. Biochem. 1976,72,359-365. 161 Van Jaarsveld, P. P., van der Walt, B. J. and Le Roux, C. H., Anal. Biochem. 1976,75,363-373. [71 Southern, E. M., Anal. Biochem. 1979,100,104-318.
Electrophoresis 199 1,12, YO-93
181 Carreira, L. H., Carlton, B. C., Bobbio, S. M., Nagao, R. T. and Meagher, R. B., Anal. Biochem. 1980,106,455-468. 191 Hediger, M., Anal. Biochem. 1984,142,445-454. [ 101 Hediger, M., Anal. Biochem. 1986,159,280-286. [ I 11 Curioni, A,, Peruffo, A. D. B. and Nuti, M. P.,Electrophoresis 1988, 9,327-330. 1121 Hjerten, S., Jerstedt, S. and Tiselius, A., A n d . Biochem. 1969, 27, 108-129. [ 131 Stegrnann, H., in: Radola, B. J. (Ed.), Electrophoresis '79, Walter de Gruyter, Berlin 1980, pp. 571-582. [I41 Yoshida, K., Anal. Biochem. 1983,129,37-45. 1151 Carpenter, H. C., Skerritt, J. H., Wrigley, C. W. and Margolis, J., Electrophoresis 1986, 7,22 1-226. [161 Laernmli, U. K., Nature 1970,227,680-685.
Preparative electrophoresisof aggregated bacterial proteins
93
1171 Erler, W., Arch. Exptl. Vet. Med. 1973,27, 321-326. I 181 Feist, H., Flossmann, K. D. and Erler, W., Arch. Exptl. Vet. Med. 1976,30,49-57. [191 Burnette, W. N., Anal. Biochem. 1982,112, 192-203. [201 Tirnoney, J. F. and Trachrnan, J., ZnJ Immun. 1985,48,29-34. 1211 Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J.,J. Biol. Chem. 1951,193,265-275. [221 White, T. G. and Vervey, W. F., ZnJ Zmmun. 1970, I , 387-393. 1231 Groschup, M., Thesis, Fachbereich Veterinaermedizin of the JustusLiebig-Universitaet Giessen, Germany, 1988. [241 Galan, J. E. and Tirnoney, J . F., ZnJ Zmmun. 1985,47, 623-628. [251 Boschwitz,J.,Groschup,M. andTirnoney,J.F.,Cornell Vet. 1991,in press.
Electrophoresis 1991, I2,90-93
M. H. Groschup et al.
1211 Flynn, T. G., Biochem. Pharm. 1982,31,2705-2712. [221 Markus, H. B., Raducha, M. and Harris, H.,Biochem. Med. 1983,29, 31-45. 1231 Sato, S. and Kador, P. F., Invest. Ophthal. Vis. Sci. 1989, 30, 1618- 1622. [241 Tanimoto, T., Fukuda, H. and Kawamura, J., Chem. Pharm. Bull. 1983,31,2395-2403. [251 Schade, S. Z., Early, S. L., Williams, T. R., Kezdy, F. J., Heinrikson, R. L., Grimshaw, C. E. and Doughty, C. C.,J. Biol. Chem. 1990,265, 3628-3635. [261 Carper,D.,Nishimura,C.,Shinohara,T.,Dietzchold,B., Wistow,G., Craft, C., Kador, P. and Kinoshita, J. H., FEBS Lett. 1987, 220, 209-213. [271 Bohren, K. M., Bullock, B., Wermuth, B. and Gabbay, K. H.,J. Bid. Chem. 1989,264,9547-9551.
Martin H. Groschup Jeff Boschwitz John F. Timoney New York State College ofveterinary Medicine, Cornell University, Ithaca, NY
1281 Garcia-Perez, A., Martin, B., Murphy, H. R., Uchida, S., Murer, H.. Cowley, B. D., Handler, J . S. and Burg, M.B., J.Biol. Chem. 1989, 264, 16 815-16 821. [291 Nishimura, C., Graham, C., Hohman, T. C., Nagata, M., Robison, W. G. and Carper, D., Biochem. Biophys. Res. Commun. 1988,153, 1051-1059. [301 Tarle, I., Skow, L. C. and Petrash, J. M., Invest. Ophthal. Vis. Sci. 1990,31, (Suppl.), 353. [311 Kador, P. F. and Sharpless, N. E., Mol. Pharmacol. 1983, 24, 521-53 1. [321 DeLucas, L. J., Bowling, E. and Petrash, J. M., Invest. Ophthal. Vis. Sci. 1987,28, (Suppl.), 87. [331 DeLucas, L. J., Dissertation, University of Alabama at Birmingham, 1981.
A convenient gel holder for preparative electrophoretic separation of aggregated bacterial proteins A simple custom-made gel holder for preparative SDS-PAGE to separate aggregated bacterial antigens is described. The gel holder fits easily into commercially available gel electrophoresis apparatus and proteins or peptides are collected in a stream of distilled water through a channel in the gel. The performance ofthe device was illustrated by the successful separation and purification of fragments of aggregated Streptococprotective antigens. Yields of cus equi M protein and of Erysipelothrix rhusio~ath~ae up to 1.2 mg per run were obtained. The purified proteins retained immunological reactivity and were of sufficient purity for amino acid compositional and sequence analysis.
1 Introduction Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used and effective technique for the separation and resolution ofproteins by their molecular weight. Attempts to scale up this method to the preparative mode have utilized separation on an analytical vertical SDS-PAGE slab gel with subsequent visualization and electroelution of the desired protein band. In commercially available “successive zone elution” analytical scale devices the eluting band is collected in a single step into dialysis tubing at the lower end of the resolving gel. However, the time to harvest, which is critical to the success of the separation, is difficult to control. In addition, the yields using this method are poor. An apparatus for true preparative electrophoresis should allow the application of greater amounts of sample with continuous elution of the separated bands from the resolving gel. A variety of apparatus, based on cylindrical gel columns with cooling systems, have been described [ 1-1 11. Such equipment is highly complex and expensive.
Correspondence: Dr. M. H. Groschup, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 18453, USA Abbreviations: PBS, phosphate buffered saline; PAGE, polyacrylamide gel electrophoresis; SDS,sodium dodecyl sulfate
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Flat gel systems exhibit better heat dissipation due to their greater surface area [12-141. A flat gel modification device described recently [ 15I permits continuous collection of bands from the lower end of the gel following a scaled-up analytical run. This paper describes the successful purification of components of aggregated bacterial antigens of Streptococcus equi and Erysipelothrix rhusiopathiae by using a simple custom-made vertical flat-bed gel holder designed for preparative electrophoresis.
2 Methods 2.1 The preparative gel apparatus
The gel holder (Fig. 1) was designed to fit into a conventional vertical gel electrophoresis apparatus (Hoefer SE 400 “The Sturdier”) and consisted of two acrylic plates (17.6 x 16.1 x 0.45 cm) with acrylic spacers (5.5 x 11 x 0.45 cm and 5.5 x 4.6 x 0.45 cm, respectively) glued between to provide space (9 x 6.6 x 0.45 cm) for the stacking and resolving gel. A transverse horizontal channel for elution of bands during the run was positioned 4.6 cm from the bottom of the gel holder. The gels were poured using the discontinuous buffer system of Laemmli [ 161 and set up as follows. A 24 % acrylamide solution was poured below the channel to prevent the escape of proteins during the elution process. A bar (14.5 x 0.45 x 0.45 0173-0835/9l/010l-0090 %3.50+.25/0
Preparative electrophorev\ of aggregated bacterial proteins
Electrophoresis 199l , I 2 , YO-93
r-
17.6 cm
12.3
91
Electrophoresis and elution procedure
1
pq 16.1
7’
Figurel. Sketchshowing thegeiholder.Spaceforthestacking(l),resolving ( 2 ) and supporting gel (3) is provided between two Plexiglas plates glued together by four spacers. Eluting bands are harvested through a horizontal channel(4) thatisclosedattheinlet(5)andoutletsides(6)bybushings(7,7’) with polyethylene tubing (8,8‘). The gel holder is fixed by screws (9”) into clamps (9) with slots for cams (9”’) that fit into commercially available gel electrophoresis apparatus.Access to the inlet and outlet ports is via openings on the side of the clamps (9’).
The samples in a total volume of approximately 2 mL were applied to the top of the stacking gel (4 % acrylamide) and separated at 35 mA until the dye front entered the collecting channel (0 rnin). The current was then adjusted to 45 mA, the channel was rinsed for 12 rnin with distilled water (9 mL/ min) toflushoutelutedSDS, and proteins andfractions(9mL) were collected at the expected time of elution of the desired band as determined previously by running cytochrome oxidase and bovine serum albumin prestained with Remazol Blue. The approximate elution times of the 66-64 kDa band from alkaline extracts of E. rhusiopathiae was 48-62 min in the 8% polyacrylamide gel with 0.4 % SDS. The 4 I kDa band band from acid extracts of S . equi CF32 eluted after 12-25 min from the 8 % polyacrylamide gel with 0.03 % SDS. All collected fractions were lyophilized (Speedvac Concentrator System, Savant, NY), resolubilized in 100 pL distilled water and analyzed (20 pL) by SDS-PAGE (12 % or 10 %).
2.4 Immunoblotting
Proteins were electrophoretically transferred to nitrocellulose cm) coated with polytetrafluoroethylene was inserted from the [ 191 and immunoblotted. Immunoblots were prepared with port at the side to provide space for the channel between the porcine antiserum to E. rhusiopathiae (WDT, Hoyerhagen, supporting and the resolving gels. The holes at the sides were Germany) followed by peroxidase conjugated protein A (Bioquickly plugged by plastic bushings and the acrylamide solu- Rad, Munich) or rabbit antiserum to S. equi M protein [201 tion was poured to a level of 1 mm above the bar to com- followed by peroxidase conjugated goat anti-rabbit serum pensate for contraction during polymerization. The resolving (Zymed, San Francisco, CA), respectively. Immunoreactive gel of the desired polyacrylamide and SDS concentrations bands were visualized with 4-chloro- 1-naphthol as substrate. was then poured to a height of 6 cm, overlayered with distilled Fractions containing the desired band were pooled and prowater, and allowed to polymerize at room temperature for at tein yields determined by the method of Lowry et al. [21]. least 4 h. Finally, the stacking gel (4 % polyacrylamide) was added. Before removal from the channel, the bar was pushed back and forth several times, using a stick to dislodge scraps of 3 Results and discussion polymerized polyacrylamide. The channel was then flooded with tank buffer (0.1 % SDS in 0.75 M Tris, pH 8.6), air 3.1 Preparative electrophoresis bubbles were driven out and the outlets closed by two plastic bushings with holes drilled through them to accommodate E. rhusiopathiae is an important gram positive pathogen of polyethylene tubing. The inlet side was connected to a distilled pigs and turkeys and carries a protective antigen in the form water reservoir. Fractions of 9 mL were collected into of a glycolipoprotein complex of approximately 200 kDa disposable tubes via a tube from the outlet side. During collec- molecular mass which can separateinto smaller subunits 1221. tion, distilled water or tank buffer diluted 1:100 in distilled Efforts to purify these subunits from extracts by conventional water was flushed through the channel at a rate of 9 ml/min, chromatography resulted in a dramatic decrease of their prothe flow rate being controlled by raising or lowering the reser- tective ability. Our work, using the gel holder and procedure voir attached to the entry port. described above, represents the first successful purification of a protective subunit (66-64 kDa) from alkaline extracts ofE. rhusiopathiae in which protective activity in a mouse protection model was retained (Groschup et al., unpublished data). 2.2 Sample preparation Yields of 0.2-1.2 mg per run were achieved (Fig. 2). An alkaline (0.01 M NaOH) extract 171 of E. rhusiopathiae strain T 28 cells, grown in an overnight (20 h/37 “C) culture Hot acid extraction is a commonly used method to obtain M (500 ml) 1181, was concentrated by ultrafiltration to 1 mL protein from S . equi [201, but it degrades this protein into volume, mixed with an equal volume of double-strength several hydrophobic fragments which tend to aggregate in Laemmli sample buffer [ 161and incubated for 30min at 56 OC aqueous solution. Attempts at purifying individual fragments before application to the gel holder. The SDS and poly- using ion exchange 1231, reverse phase (Timoney et al., unacrylamide concentrations were 0.4 % and 8 %, respectively. published data) and gel sieve exclusion chromatography 120, In the case of S . equi strain CF32, bacterial cells from one L 241 have been unsuccessful due to elution of the fragments as Todd Hewitt broth were extracted by hot acid (HCl, pH 2.5, aggregates in a single peak. Preparative electrophoresis using 95 “C, 10 min). Proteins that adsorbed to hydroxylapatite the custom built gel holder enabled the purification of a 4 1kDa (Bio-Rad, San Francisco, CA) in 10 mM sodium phosphate fragment of S . equiM protein (Fig. 3). This fragment has been buffer, pH 7.2, were eluted with 0.5 M sodium phosphate, pH implicated in protective nasopharyngeal mucosal antibody 9.0, desalted, and loaded in the gel holder containing 8 % responses [241. Quantities of approximately 50- 100 pg purified fragment per run were obtained and were suficient for anpolyacrylamide and 0.03 % SDS resolving gel.
92
Electrophoresis 1991,12, 90-93
M. H. Groschup et al.
Figure 2.Immunoblot offractionsfrom apreparative electrophoresisrun of an alkaline extract of E. rhusiopathiae. One fifth of the harvested fractions (9 mL) were lyophilized and resolubilized in distilled water, separated by SDS-PAGE(I0 %),transferredto nitrocelluloseandimmunoblotted with a porcine antiserum to E. rhusiopathiae (1 :100 in phosphate-buffered saline (PBS) -0.1 Yo Tween 20), followed by protein A (1:1000 in PBS-0.1 % Tween 20) and 4-chloro-1-naphthol as substrate. The numbers on top are the elution times of the fractions in minutes, those on the side are the approximate molecular masses x 1000(kDa). An immunoblot of untreated extract is shown on the right.
Figure3. Immunoblot offractionsfrom apreparativeelectrophoresisrunof a hot acid extract of S. equi. Aliquots of the harvested, lyophilized and resolubilized fractions were separated by SDS-PAGE (10 %), transferred to nitrocellulose and immunoblotted with a rabbit antiserum to S. equi M protein (1 :200 in PBS-0.1 % Tween 20), goat anti-rabbitconjugated peroxidase (1:1000 in PBS-0.1 %Tween 20) and 4-chloro-I-naphthol as substrate. The numbers on top are the elution time of the fractions in minutes, those on the side the approximate molecular weight x 1000(kDa). An immunoblot of nonfractionated extract is shown on track (A).
tiserum production and amino acid compositional and sequence analysis [251.
minimized exposure time to the acrylamide and shortened the fractionation time. Instead of a bar, a transverse elution channel can also be formed by layering the resolving gel on top of a dense sucrose solution during the pouring process. However, an additional step is necessary to thoroughly wash the channel free of sucrose. Either method of forming the channel gave good results, the channel being free of debris and patent during the run. Adhesion of acrylamide to the bar was not a problem at concentrations of acrylamide between 7 and 18 %.The high acrylamide concentration of the supporting gel and the high flow rate (9 mL/min) through the harvesting channel enhanced both separation and yields. SDS is washed off before fractions were collected. If necessary, SDS residues in the harvested band can easily be removed by column chromatography on Extracti-GelTMD (Pierce, Rockford, IL) detergent removing gel. Antigen peaks eluted at highly predictable times during different runs and thus reduced the number of fractions that had to be collected. The recovery rate was 25-40 % when ovalbumin was separated from bovine albumin and cytochrome oxidase. Heat dissipation during runs was adequate due to the favorable volume-to-surface-area ratio of the gel. Additional cooling, if necessary, could easily be provided by attaching a cooling plate to either the freely accessible front or back of the gel holder surface. Because of the relatively narrow width of the gel “smile” effects were negligible. This is an advantage of the custom-made gel holder compared with commercially available vertical gel apparatus fitted with ported spacer kits, where curvature of the bands can result in contamination during elution. The short length of the harvesting channel reduces the time available for the migrating protein to enter the supporting gel during harvesting. This potential source of protein loss is therefore minimized, compared to that which occurs in a wider gel apparatus.
4 Concluding remark In conclusion, preparative electrophoresis with the inexpensive custom-made gel holder provides a simple solution to the separation of aggregated surface components with retention of immunological activity, such as described from E . rhusiopathiae and S . equi.
Supported in part by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to M.H.G.). Thefinancia1 support of the Zweig Memorial Fund is also gratefully acknowledged. Received May 23, 1990
3.2 Construction and operation of the gel holder The gel holder was constructed to fit easily into commercially available gel electrophoresis apparatus such as “The Sturdier”. Leakage from the upper electrode buffer reservoir was prevented by the action of cams inserted in slots in the clamps to compress the rubber gasket between the gel holder and the upper buffer tank. The gel holder could be constructed of glass or, as in the apparatus presented, of Plexiglas. Polycarbonate (lexan) plates were found to be unsuitable because this material blocks the polymerization of polyacrylamide, probably by release of surface-fixed oxygen. Relatively low acrylamide concentrations in the resolving gel were utilized for separating the components of each bacterial extract. This
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