APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1976, p. 288-293 Copyright ©D 1976 American Society for Microbiology
Vol. 32, No. 2 Printed in U.S.A.
Biochemistry of Vibrio cholerae Virulence: Purification of Cholera Enterotoxin by Preparative Disc Electrophoresis A. CARTER LEWIS,' STEPHEN H. RICHARDSON,* AND BARBARA SHERIDAN2 Department of Microbiology, The Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103
Received for publication 26 February 1976
Procedures for cholera enterotoxin purification previously developed in this labarotory were not applicable to large-scale purification, and these methods resulted in low yields of pure toxin. An efficient scheme has been developed whereby pure cholera enterotoxin can be obtained from 6 to 8 liters of culture supernatant fluid. This method consists of concentration by membrane ultrafiltration followed by gel filtration and cation-exchange chromatography. Pure cholera enterotoxin of high biological potency was obtained after a final step of preparative acrylamide gel electrophoresis. The degree of purity of the toxinantigen as well as its biological activity were determined at various steps of purification. This alternate technique for purification is offered because of the widespread interest in cholera enterotoxin as a specific stimulator of adenyl cyclase. A number of investigators have described methods for obtaining exoenterotoxin from Vibrio cholerae. Coleman et al. (3) were able to isolate a "type 2" ileal loop toxin when they fractionated concentrated peptone supernatant fluids on Sephadex G-200 followed by salt gradient elution from diethylaminoethyl-Sephadex. The toxin was reported to be homogeneous when examined by immunoelectrophoresis, immunodiffusion, and thin-layer chromatography techniques. Finkelstein and LoSpalluto (5) described a method which involved sequential filtration of cell-free supernatants through membranes with graded pore sizes and chromatography on Sephadex, agarose, and diethylaminoethyl-Sephadex. This yielded an enterotoxin which was homogeneous when examined by Ouchterlony, ultracentrifugal, immunoelectrophoretic, and disc electrophoretic techniques. Richardson et al. (11) and Richardson and Noftle (12) developed a technique involving a selective toxin precipitation from cell-free supernatant fluids with dextran sulfate and ammonium sulfate. Further purification employed gel filtration and chromatography on diethylaminoethyl-Sephadex. A protein that exhibited both skin permeability and enterotoxic activities, that was virtually free of contaminating ' Present address: Department of Internal Medicine, Infectious Disease Section, Louisiana State University Medical Center, New Orleans, LA 70112. 2 Present address: Department of Anatomy, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA 23298.
somatic antigens, and that appeared homogeneous by immunodiffusion, immunoelectrophoresis, and disc electrophoresis was obtained. Although this procedure yielded milligram quantities of high-purity cholera enterotoxin, total recovery averaged only about 15%. Rappaport et al. (9) have recently described a method for selectively concentrating the enterotoxin of V. cholerae from cell-free supernatant fluids by co-precipitation with hexametaphosphate. The toxin was further purified by ad-
sorption on aluminum hydroxide powder. Removal of most somatic antigens and other contaminants was achieved by lyophilization and treatment with activated carbon. Toxin purified by this procedure was characterized by immunodiffusion, acrylamide gel electrophoresis, ultraviolet spectrum, and approximately 50% recovery. During the past several years, we have concentrated on developing procedures to obtain larger quantities of pure cholera enterotoxin within the limits of the equipment and resources available to us. The previously mentioned procedure employing dextran sulfate was not applicable to large-scale purification because of the low recovery. The technique reported here results in a 50 to 60% recovery of starting biological activity in a highly purified state and is applicable to multiliter volumes. We are offering this alternate technique for purification because of the widespread interest in enterotoxin as a specific stimulator of adenyl cyclase.
288
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PURIFICATION OF CHOLERA ENTEROTOXIN
MATERIALS AND METHODS
Microorganisms. Inaba strain 569B of Vibrio cholerae was used throughout this study as the toxin-producing microorganism. Maintenance of stock cultures and conditions of growth were as previously described (10). Assays. The toxin-antigen content of fractions obtained from this purification procedure was determined in three ways: passive hemagglutination inhibition (PHI) as described by Callahan et al. (2), radial immunodiffusion in agar, and limit of flocculation (Lf). The radial immunodiffusion and Lf determinations were modifications of those described by Finkelstein and LoSpalluto (5). The radialimmunodiffusion technique employed "immuno-plates" (Hyland, Div. of Travenol Laboratories, Inc., Costa Mesa, Calif.) containing monospecific antiserum (2). The antiserum was diluted 1:150 in 4 ml of melted and cooled 1% Nobel special agar with 1% sodium azide. Wells were cut with a stainless-steel cutter, and the agar was removed by suction through a sawed-off hypodermic needle. The wells were 1.88 mm in diameter; two patterns of five peripheral wells were made in each plate, and the wells were filled with 8 Al of test material. The assays were performed in quadruplicate, and the plates were placed in a humid chamber for 16 to 18 h at room temperature. The diameters of the zones of precipitation were determined with the aid of a microscope fitted with an ocular micrometer. The mean diameters were compared with a standard curve for the determination of micrograms of antigen per milliliter. The reference antigen preparation was standardized by the PHI assay. In the Lf procedure, 0.025 ml of monospecific antiserum, heat inactivated at 56°C for 30 min and diluted 1:5 in phosphatebuffered saline, was added to a series of Microtiter wells (V-plate) containing 0.025 ml of purified toxinantigen standards ranging from 1.18 to 4.75 gg. The serum antigen mixtures were mixed on a Vortex shaker, pulled into capillary tubes (1.1- to 1.2-mm inside diameter), and observed by transmitted oblique illumination at room temperature to determine the Lf, or most rapidly flocculating mixture. The first tube of flocculate was found to contain 534.4 Lf units per ml of undiluted serum. The standardized serum was then used to determine the number of Lf units present in various antigen-containing fractions obtained throughout the purification scheme. The biological activity of the various fractions was determined by a modification of the limit-ofbluing system of titration of cholera permeability factor toxin, as described by Craig (4). Standardization of the monospecific antiserum was carried out as follows. A 0.5-ml portion of twofold serial dilutions of heat-inactivated antiserum in phosphatebuffered saline was added to 0.5 ml of the standard toxin preparation (2,000 bluing doses/ml) in test tubes (13 by 100 mm). The tubes were mixed well and incubated at 37°C for 60 min. After incubation, 0.1 ml of each antigen-antibody mixture was injected in duplicate into the shaved backs of two New Zealand white rabbits. At 18 to 20 h later 0.5 ml of
289
5% Evans Blue dye was injected intravenously 1 h prior to the time of reading. The dilution of antibody that yielded a blue area measuring 4 mm in diameter was considered to be the end point and was assigned a value of 1 standard antitoxin unit. Our antitoxin unit is therefore defined as that amount of antibody which will reduce 100 bluing doses to a 4mm blue lesion. The standard antiserum was found to contain 25,000 antitoxin units/ml and was employed in determining the limit-of-bluing dose of toxin present in the antigen-containing fractions obtained during the purification procedure. The assay was as described above, except that a known amount of antitoxin was added to varying amounts of test material. Our limit-of-bluing dose of toxin is defined as that amount of permeability factor toxin which will evoke a lesion of vascular permeability 4 mm in diameter in the presence of 1 unit of antitoxin. Protein was determined by the method of Lowry et al. (8) using crystalline bovine serum albumin as a standard. Fermenter culture. The standard medium for large-scale toxin production was TRY, which consisted of, in grams per liter of 5 mM tris(hydroxymethyl)aminomethane (Tris)-maleate buffer (pH 7.5): NaCl, 2.5; KCl, 2.5; Na2HPO4, 0.2; Casamino Acids (Difco Laboratories, Detroit, Mich.), 10.0; yeast extract (Difco), 0.05; and glycerol, 0.5, plus 1 ml/liter of a mixture of 5% MgSO4, 0.5% MnCl2 * 4H20, and 0.5% FeCl3 in 0.4% nitrilotriacetic acid. A seed culture was prepared by inoculating 80 ml of TRY medium, contained in a 300-ml flask, with 0.8 ml (108 colony-forming units) of a saline suspension of cells from an overnight Columbia agar (BBL, Cockeysville, Md.) slant incubated at 37°C. The seed culture was incubated for 6 h at 25°C at 250 rpm on a rotary shaker (Psycrotherm; New Brunswick Scientific Co., New Brunswick, N.J.), and the entire contents were used as inoculum for 7.5 to 8.0 liters of medium in the fermenter (Microferm; New Brunswick Scientific Co.). Fermenter cultures were incubated at 25°C and 240 rpm under continuous aeration with a filtered airflow of 1,000 ml/min. Foam created by aeration was automatically controlled with an aqueous suspension of silicone. Cultures were grown for 16 h to achieve maximum growth and toxin-antigen production (2). Preparation of crude toxin. All procedures for concentrating the supernatant fluid from fermenter cultures, as well as purification steps, were carried out at 4°C unless otherwise stated. The organisms were removed by centrifugation at 20,000 x g for 20 min, and the supernatant fluid was filter sterilized by passage through a prewashed 0.45-,Mm membrane filter (Millipore Corp., Bedford, Mass.). Merthiolate was added to the culture filtrate in a sufficient quantity to reach a bacteriostatic level (1:20,000). The filtrate was then concentrated 20-fold by pressure ultrafiltration in a model TCID ultrafiltration cell (Amicon Corp., Lexington, Mass.) using a UM10 Diaflo membrane. The effluents from all concentration steps were monitored for the presence of antigen by a passive hemagglutination test (2) before discarding.
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APPL. ENVIRON. MICROBIOL.
The concentrated retentate was then passed the toxin-antigen during the purification procedure, through an XM-100 Diaflo membrane to remove ma- samples were subjected to analytical disc electroterial of a molecular size greater than 100,000. The phoresis using acrylamide gel electrophoresis. PorXM-100 retentate was homogenized and subjected to tions containing 50 to 200 mg of protein from each ultracentrifugation at 100,000 x g for 2 h. The pellet step of purification, a small amount of sucrose, and was resuspended and washed twice in phosphate- tracking dye were applied to a 5.0% acrylamide gel buffered saline, pH 7.0, and the washings were slab. The gel was prepared in 0.1 M Tris-boratepooled with the XM-100 ultrafiltrate. The retentate ethylenediaminetetraacetate buffer, pH 8.5, and and washings were concentrated to a final volume of equilibrated by prerunning at 100 mA for 30 min. 30 ml on a UM-10 Diaflo membrane. This procedure The samples were subjected to electrophoresis at 50 is necessary to recover an additional 20 to 40% toxin- mA until the dye entered the gel, and the electroantigen "trapped" by the high-molecular-weight li- phoresis was then increased to 125 mA for the repopolysaccharide. mainder of the run. The gel slab was removed from Toxin purification. The concentrated preparation the cell and placed in 12% trichloroacetic acid for 2 was applied to a column (5 by 60 cm; Pharmacia Fine h. After fixation of the protein the gel was washed Chemicals, Inc., Piscataway, N.J.) packed with with tap water and placed in 0.25% Coomassie blue Bio-Gel agarose 0.5M (Bio-Rad Laboratories, Rich- for 5 to 6 h. The gel was then destained in an mond, Calif.) equilibrated with 5 mM sodium phos- electrophoretic destainer with methanol-water-glaphate buffer, pH 7.2, containing merthiolate cial acetic acid (5:5:1). The gels were stored in 5% (1:20,000). Elution with the equilibrating buffer at glycerine. a flow rate of 60 ml/h removed the larger-molecularsize lipopolysaccharide as well as the smallerRESULTS molecular-size medium constituents. These and The various steps involved in the purification subsequent column fractions were monitored continuously by absorbance at 280 nm with a Beckman of the cholera exoenterotoxin on a large-scale DB spectrophotometer (Beckman Instruments, Inc., basis are demonstrated by the flow diagram in Fullerton, Calif.) equipped with a flow cell. The Fig. 1. The steps involving membrane ultrafiltoxin-antigen fractions, recognized by the passive tration and ultracentrifugation deal with conhemagglutination assay, were pooled, adjusted to centrating 7 to 8 liters of culture filtrate to pH 6.5 with 5 mM monobasic sodium phosphate, approximately 30 ml. There is little purification and applied to a Bio-Gel AG50W cation-exchanger column (2.8 by 80 cm) equilibrated with 5 mM so- of the toxin-antigen during these steps, except dium phosphate buffer, pH 6.5. The toxin-antigen for the removal of the high-molecular-weight was eluted with 10 mM dibasic sodium phosphate material which is discarded in the washed peland concentrated to approximately 15 ml employ- let after ultracentrifugation. In addition, small ing a UM2 membrane. The concentrated toxin- amounts of low-molecular-weight material are antigen was subjected to preparative gel acrylamide discarded in the ultrafiltrates. electrophoresis using a Canalco Prep-Disc (Canal Gel filtration. The additlon of the concenIndustrial Corp., Rockville, Md.) equipped with a constant-voltage Buchler power supply (Buchler InCRUDE CULTURE FILTRATE struments Div., Nuclear-Chicago Corp., Fort Lee, 7.5 LITERS N.J.). Purification of the toxin-antigen was accomCONCENTRATED plished by passage through a 3.0-cm length of 4.0% UM-IO acrylamide stacking gel and through a 0.7-cm RETENTATE length of 7.0% acrylamide separating gel. The stackULTRAFILTRATE XM-100 DISCARD ing and separating gels were prepared in 0.1 M TrisRETENTATE 100,000 x g borate buffer, pH 8.5 and 9.5, respectively. After ULTRAFILTRATE PELLET WASH 2X PBS pH 74 polymerization of the gels with ammonium persul,| ~~~WASH INGS fate the apparatus was assembled, and the electrode WASHED PELLET chambers were filled with 0.06 M Tris-borate buffer, DISCARD CONCENTRATED pH 8.5. The column was then cooled to 4°C by circuUM-IO lating ice water through the cooling compartments. ULTRAFILTRATE RETENTATE A few crystals of sucrose were added to the sample to DISCARD BIO-GEL A-0.5 M permit layering on top of the stacking gel. A small r ANTIGEN FRACTIONS LIPOPOLYSACCHARIDE amount of tracking dye was added to monitor the BIO-GEL AG 50W LOW MW MATERIAL progress of the sample through the gel. Electropho| DISCARD EQUILIBRATING BUFFER EWTION ANTIGEN EWTED resis was started at 3.0 mA and continued until the DISCARD CONCENTRATED UM-2 sample entered the stacking gel. The current was then increased to 7.5 mA for an additional 5 h. RETENTATE ULTRAFILTRATE PREPARATIVE GEL DISCARD Elution was carried out with 0.1 M Tris-borate ELECTROPHORESIS buffer, pH 9.5. A peristaltic pump (Buchler InstruNON-ANTIGEN FRACTIONS ments) was used to maintain a flow rate of 40 ml/h. PURIFIED TOXIN ANTIGEN DISCARD The antigen content of the fractions was determined by the passive hemagglutination inhibition assay: FIG. 1. Flow diagram for cholera enterotoxin puAfter the removal of contaminating material from rification from TRY medium.
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291
from the toxin in this step, since the overall net charge of the two is so close at the pH employed for elutions. Preparative acrylamide electrophoresis. The pooled and concentrated fractions were then subjected to preparative gel acrylamide electrophoresis. A typical elution profile is shown in Fig. 3. The optical density of each fraction was plotted at both 280 and 260 nm to help locate the antigen. Titrations of the fractions were determined by the PHI assay and are plotted as micrograms of antigen per milliliter. Antigen elutes just after the tracking dye and unpolymerized acrylamide, which has a high 260-nm absorbance. Antigen elution begins slowly, reaches a plateau, and then emerges as a rather sharp peak. The fractions containing homogeneous toxin-antigen have absorbancy at 280 nm/absorbancy at 260 nm ratios approaching 2. A photograph of selected fractions and the starting material which were subjected to analytical gel electrophoresis can be seen in the upper left corner of Fig. 3. The antigen elution profile as well as the position of the protein bands on the analytical gel suggest the possibility of several species of toxin-antigen. This is supported by the fact that the antibody used in the PHI assay was made using material eluted in the sharp peak as the immunogen. The antigen fractions eluted first possess little or no biological activity (data not presented) but do react in the PHI assay. This may indicate that these species or subunits of toxin-antigen possess a high proportion of antibody reactive sites which are not biologically active. Previous experiments have shown that the heavy band seen in the concentrated starting material (Fig. 3) at the top of the analytical gel possesses no permeability factor activity but gives a reaction of identity with the purified toxin when reacted with monospecific antibody. In analogy to the data of Finkelstein and LoSpalluto (5), this material is toxoid. Elution of the naturally occurring toxoid from the preparative column can be accomplished by continuing electrophoresis for an additional 18 to 20 h. A more efficient means of obtaining purified toxoid is to reduce the percentage of acrylamide in the stacking gel from 4.0 to 3. 0%. Under these conditions the toxin peak and the tracking dye overlap, but the toxoid can be eluted in a 10- to 12-h period. Table 1 gives the percentage of recovery of micrograms of antigen and biological activity 4 12 20 28 36 44 52 60 68 76 for each step of the purification scheme. The FRACTION (5 ML) percentage of recovery is expressed as microculture fil- grams of antigen based on the PHI assay and as FIG. 2. Elution profile of TRY crude limit-of-bluing doses, with the activity of the trate from agarose 0.5M column. The toxin-antigen unconcentrated crude culture filtrate serving was determined by PHI.
trated toxin to the Bio-Gel agarose column (Fig. 1) is the first step in the removal of contaminating material. A typical elution profile from the agarose column can be seen in Fig. 2; the optical density at 280 nm and the micrograms of antigen per milliliter, determined by the PHI, are plotted for each 5-ml fraction. It is apparent that the material eluted in the column void volume contains very little antigen and represents molecules of 500,000 molecular size or greater (e.g., residual lipopolysaccharide). The toxin-antigen was eluted in the second peak and contains some colored material which apparently has a molecular size similar to the antigen. The dark-colored material eluted at the end of the column volume would have a molecular size of 5,000 to 10,000 and, for the most part, probably represents the low-molecular-weight medium constituents. Ion-exchange chromatography. The antigen-containing fractions from the agarose column were pooled and adjusted to pH 6.5 with 5 mM monobasic sodium phosphate. This material was added to the Bio-Gel AG50W cationexchanger column and eluted with 2 bed volumes of the equilibrating buffer or until the optical density reached zero. The antigen was eluted from the cation-exchanger with 10 mM dibasic sodium phosphate, pH 8.5. The ion-exchange step was carried out at room temperature, because the more concentrated fractions precipitate at lower temperatures at this pH. The pooled antigen fractions were concentrated on a UM-2 membrane to a total volume of 15 ml. An advantage of using the Bio-Gel cationexchanger is that it can be reconstituted in situ and can therefore be used for several purification cycles without having to repack the column. There is little separation of the toxoid
292
APPL. ENVIRON. MICROBIOL.
LEWIS, RICHARDSON, AND SHERIDAN
S20
W
2~~~~~~~~~~~~~~ / o~=~\\ N V500W
Nb'
.200
.~~~~~~~~~~~~~ .300
0 150
too1
.200
.100/100 2
+
(.
%
10
12 1X
16
18
20
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24
FRArCTION (1 ML)
26 28
30
32
3+34
381
40
e 4
FIG. 3. Elution profile of concentrated toxin-antigen from preparative gel acrylamide column. Antigen levels were determined by PHI. In upper left corner is a photograph of selected fractions and starting material (SM) which were subjected to analytical gel electrophoresis. TABLE 1. Summary of cholera toxin purification Material
CF'
Vol (ml)
Total protein (mg)
PHI 47 707
Antigen (,ug/ml)a Lf
RD
of Lb/,ug proteinb
Recovery_(%) PHI Lb
54 53 2.75 100 100 7,400 4,839.60 XM-100 495 712 690 3.07 100 100 4,515.00 UM-10 32.5 3,848.00 3.80 100 99 10,688 12,024 9,400 A-0.5M 670 385.92 396 427 26.12 76 75 430 AG50W 20 295.20 72 75 32.50 12,492 12,220 13,400 PA" 15 151.86 56 49.48 43 9,996 10,650 11,250 a Toxin antigen was determined by passive hemagglutination inhibition (PHI), limit of flocculation (Lf), and radial immunodiffusion (RD). Biological activity expressed as limit-of-bluing (Lb) doses. Crude culture filtrate. "Preparative acrylamide.
as 100%. Again, the first three steps of the purification scheme are concentration procedures with little or no loss of either antigen or biological activity. The 25% drop in both biological activity and antigen could not be detected in the 500,000-molecular-weight peak from the agarose column. This might be explained on the basis that the lipopolysaccharide is attaching to the antigen in such a way that the antigenic and/or biological sites are covered. This could in turn interfere with the detection of the
toxin-antigen in the various assays. The AG50W cation-exchange step appears to remove contaminating substances with a minimum loss of antigen or biological activity. The preparative acrylamide electrophoresis step results in an apparent loss of antigen, because only purified toxin is eluted. The loss of additional biological activity suggests some conversion of toxin to toxoid during fractionation by polyacrylamide gel electrophoresis. The final product had 56% of the total biological activity
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PURIFICATION OF CHOLERA ENTEROTOXIN
of the crude culture filtrate and was 18 times as toxic on a microgram of protein basis. DISCUSSION The specific activity of the purified toxin prepared by the technique described here ranges from 5 to 6 bluing doses per nanogram of toxinantigen, which is consistent with earlier reports from this laboratory (11, 12) as well as those from Finkelstein's laboratory (5, 6). In a more recent report, however, Finkelstein and LoSpalluto (7) state that crystallized cholera toxin has a specific activity of 20 bluing doses/ ng. Accepting this latter figure as "pure" would suggest that our final product is either heavily contaminated with other proteins or that the toxin as isolated is largely (two-thirds) biologically inert. There is no evidence of contaminating protein even on grossly overloaded analytical gel slabs. However, the diffuse multibanded appearance of some of the preparative gel fractions suggests that dissociation and reassociation of subunits may be occurring during electrophoresis. This could result in creation of inactive aggregates which would manifest themselves in lowered specific activity of the final product. The relatively long exposure to diluting buffers and charged conditions may also have an adverse effect on the toxin's biological activity without a concomitant reduction in immunochemical reactions. In summary, we have developed a method for the purification of choleragenic toxin from multiliter culture filtrates with 50 to 60% recovery of the starting biological activity. The purification scheme consists of concentration, gel filtration, ion-exchange chromatography, and preparative acrylamide gel electrophoresis as the final step. Although this procedure does not give greater yields than Finkelstein's technique, it does offer an alternative approach to procurement of reasonable amounts of purified toxin.
293
During the completion of this work, a chemically defined medium for production of cholera exoenterotoxin was formulated in our laboratory (1, 2). We have since adapted this defined medium to fermenter cultures and will present a purification scheme employing this chemically defined system in a future report. LITERATURE CITED 1. Callahan, L. T., III, and S. H. Richardson. 1973. Biochemistry of Vibrio cholerae virulence. III. Nutritional requirements for toxin production and the effects of pH on toxin elaboration in chemically defined media. Infect. Immun. 7:567-572. 2. Callahan, L. T., III, R. C. Ryder, and S. H. Richardson. 1971. Biochemistry of Vibrio cholerae virulence. II. Skin permeability factor/cholera enterotoxin production in a chemically defined medium. Infect. Immun. 4:611-618. 3. Coleman, W. H., J. Kaur, M. E. Iwert, G. J. Kasai, and W. Burrows. 1968. Cholera toxins: purification and preliminary characterization of ileal loop reactive type 2 toxin. J. Bacteriol. 96:1137-1143. 4. Craig, J. P. 1966. Preparation of the vascular permeability of Vibrio cholerae. J. Bacteriol. 92:793-795. 5. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Pathogenesis of experimental cholera-preparation and isolation of choleragen and choleragenoid. J. Exp. Med. 130:185-202. 6. Finkelstein, R. A., and J. J. LoSpalluto. 1970. Production of highly purified choleragen and choleragenoid. J. Infect. Dis. 121:S63-S72. 7. Finkelstein, R. A., and J. L. LoSpalluto. 1972. Crystalline cholera toxin and toxoid. Science 175:529-530. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Rappaport, R. S., B. A. Rubin, and H. Tint. 1974. Development of a purified cholera toxoid. I. Purification of toxin. Infect. Immun. 9:294-303. 10. Richardson, S. H. 1969. Factors influencing in vitro skin permeability factor production by Vibrio cholerae. J. Bacteriol. 100:27-34. 11. Richardson, S. H., D. G. Evans, and J. C. Feeley. 1970. Biochemistry of Vibrio cholerae virulence. I. Purification and biochemical properties of PF/cholera enterotoxin. Infect. Immun. 1:546-554. 12. Richardson, S. H., and K. A. Noftle. 1970. Purification and properties of permeability factor/cholera enterotoxin from complex and synthetic media. J. Infect. Dis. 121:S73-S79.
Vol. 32, No. 2 Printed in U.S.A.
Biochemistry of Vibrio cholerae Virulence: Purification of Cholera Enterotoxin by Preparative Disc Electrophoresis A. CARTER LEWIS,' STEPHEN H. RICHARDSON,* AND BARBARA SHERIDAN2 Department of Microbiology, The Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103
Received for publication 26 February 1976
Procedures for cholera enterotoxin purification previously developed in this labarotory were not applicable to large-scale purification, and these methods resulted in low yields of pure toxin. An efficient scheme has been developed whereby pure cholera enterotoxin can be obtained from 6 to 8 liters of culture supernatant fluid. This method consists of concentration by membrane ultrafiltration followed by gel filtration and cation-exchange chromatography. Pure cholera enterotoxin of high biological potency was obtained after a final step of preparative acrylamide gel electrophoresis. The degree of purity of the toxinantigen as well as its biological activity were determined at various steps of purification. This alternate technique for purification is offered because of the widespread interest in cholera enterotoxin as a specific stimulator of adenyl cyclase. A number of investigators have described methods for obtaining exoenterotoxin from Vibrio cholerae. Coleman et al. (3) were able to isolate a "type 2" ileal loop toxin when they fractionated concentrated peptone supernatant fluids on Sephadex G-200 followed by salt gradient elution from diethylaminoethyl-Sephadex. The toxin was reported to be homogeneous when examined by immunoelectrophoresis, immunodiffusion, and thin-layer chromatography techniques. Finkelstein and LoSpalluto (5) described a method which involved sequential filtration of cell-free supernatants through membranes with graded pore sizes and chromatography on Sephadex, agarose, and diethylaminoethyl-Sephadex. This yielded an enterotoxin which was homogeneous when examined by Ouchterlony, ultracentrifugal, immunoelectrophoretic, and disc electrophoretic techniques. Richardson et al. (11) and Richardson and Noftle (12) developed a technique involving a selective toxin precipitation from cell-free supernatant fluids with dextran sulfate and ammonium sulfate. Further purification employed gel filtration and chromatography on diethylaminoethyl-Sephadex. A protein that exhibited both skin permeability and enterotoxic activities, that was virtually free of contaminating ' Present address: Department of Internal Medicine, Infectious Disease Section, Louisiana State University Medical Center, New Orleans, LA 70112. 2 Present address: Department of Anatomy, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA 23298.
somatic antigens, and that appeared homogeneous by immunodiffusion, immunoelectrophoresis, and disc electrophoresis was obtained. Although this procedure yielded milligram quantities of high-purity cholera enterotoxin, total recovery averaged only about 15%. Rappaport et al. (9) have recently described a method for selectively concentrating the enterotoxin of V. cholerae from cell-free supernatant fluids by co-precipitation with hexametaphosphate. The toxin was further purified by ad-
sorption on aluminum hydroxide powder. Removal of most somatic antigens and other contaminants was achieved by lyophilization and treatment with activated carbon. Toxin purified by this procedure was characterized by immunodiffusion, acrylamide gel electrophoresis, ultraviolet spectrum, and approximately 50% recovery. During the past several years, we have concentrated on developing procedures to obtain larger quantities of pure cholera enterotoxin within the limits of the equipment and resources available to us. The previously mentioned procedure employing dextran sulfate was not applicable to large-scale purification because of the low recovery. The technique reported here results in a 50 to 60% recovery of starting biological activity in a highly purified state and is applicable to multiliter volumes. We are offering this alternate technique for purification because of the widespread interest in enterotoxin as a specific stimulator of adenyl cyclase.
288
VOL. 32, 1976
PURIFICATION OF CHOLERA ENTEROTOXIN
MATERIALS AND METHODS
Microorganisms. Inaba strain 569B of Vibrio cholerae was used throughout this study as the toxin-producing microorganism. Maintenance of stock cultures and conditions of growth were as previously described (10). Assays. The toxin-antigen content of fractions obtained from this purification procedure was determined in three ways: passive hemagglutination inhibition (PHI) as described by Callahan et al. (2), radial immunodiffusion in agar, and limit of flocculation (Lf). The radial immunodiffusion and Lf determinations were modifications of those described by Finkelstein and LoSpalluto (5). The radialimmunodiffusion technique employed "immuno-plates" (Hyland, Div. of Travenol Laboratories, Inc., Costa Mesa, Calif.) containing monospecific antiserum (2). The antiserum was diluted 1:150 in 4 ml of melted and cooled 1% Nobel special agar with 1% sodium azide. Wells were cut with a stainless-steel cutter, and the agar was removed by suction through a sawed-off hypodermic needle. The wells were 1.88 mm in diameter; two patterns of five peripheral wells were made in each plate, and the wells were filled with 8 Al of test material. The assays were performed in quadruplicate, and the plates were placed in a humid chamber for 16 to 18 h at room temperature. The diameters of the zones of precipitation were determined with the aid of a microscope fitted with an ocular micrometer. The mean diameters were compared with a standard curve for the determination of micrograms of antigen per milliliter. The reference antigen preparation was standardized by the PHI assay. In the Lf procedure, 0.025 ml of monospecific antiserum, heat inactivated at 56°C for 30 min and diluted 1:5 in phosphatebuffered saline, was added to a series of Microtiter wells (V-plate) containing 0.025 ml of purified toxinantigen standards ranging from 1.18 to 4.75 gg. The serum antigen mixtures were mixed on a Vortex shaker, pulled into capillary tubes (1.1- to 1.2-mm inside diameter), and observed by transmitted oblique illumination at room temperature to determine the Lf, or most rapidly flocculating mixture. The first tube of flocculate was found to contain 534.4 Lf units per ml of undiluted serum. The standardized serum was then used to determine the number of Lf units present in various antigen-containing fractions obtained throughout the purification scheme. The biological activity of the various fractions was determined by a modification of the limit-ofbluing system of titration of cholera permeability factor toxin, as described by Craig (4). Standardization of the monospecific antiserum was carried out as follows. A 0.5-ml portion of twofold serial dilutions of heat-inactivated antiserum in phosphatebuffered saline was added to 0.5 ml of the standard toxin preparation (2,000 bluing doses/ml) in test tubes (13 by 100 mm). The tubes were mixed well and incubated at 37°C for 60 min. After incubation, 0.1 ml of each antigen-antibody mixture was injected in duplicate into the shaved backs of two New Zealand white rabbits. At 18 to 20 h later 0.5 ml of
289
5% Evans Blue dye was injected intravenously 1 h prior to the time of reading. The dilution of antibody that yielded a blue area measuring 4 mm in diameter was considered to be the end point and was assigned a value of 1 standard antitoxin unit. Our antitoxin unit is therefore defined as that amount of antibody which will reduce 100 bluing doses to a 4mm blue lesion. The standard antiserum was found to contain 25,000 antitoxin units/ml and was employed in determining the limit-of-bluing dose of toxin present in the antigen-containing fractions obtained during the purification procedure. The assay was as described above, except that a known amount of antitoxin was added to varying amounts of test material. Our limit-of-bluing dose of toxin is defined as that amount of permeability factor toxin which will evoke a lesion of vascular permeability 4 mm in diameter in the presence of 1 unit of antitoxin. Protein was determined by the method of Lowry et al. (8) using crystalline bovine serum albumin as a standard. Fermenter culture. The standard medium for large-scale toxin production was TRY, which consisted of, in grams per liter of 5 mM tris(hydroxymethyl)aminomethane (Tris)-maleate buffer (pH 7.5): NaCl, 2.5; KCl, 2.5; Na2HPO4, 0.2; Casamino Acids (Difco Laboratories, Detroit, Mich.), 10.0; yeast extract (Difco), 0.05; and glycerol, 0.5, plus 1 ml/liter of a mixture of 5% MgSO4, 0.5% MnCl2 * 4H20, and 0.5% FeCl3 in 0.4% nitrilotriacetic acid. A seed culture was prepared by inoculating 80 ml of TRY medium, contained in a 300-ml flask, with 0.8 ml (108 colony-forming units) of a saline suspension of cells from an overnight Columbia agar (BBL, Cockeysville, Md.) slant incubated at 37°C. The seed culture was incubated for 6 h at 25°C at 250 rpm on a rotary shaker (Psycrotherm; New Brunswick Scientific Co., New Brunswick, N.J.), and the entire contents were used as inoculum for 7.5 to 8.0 liters of medium in the fermenter (Microferm; New Brunswick Scientific Co.). Fermenter cultures were incubated at 25°C and 240 rpm under continuous aeration with a filtered airflow of 1,000 ml/min. Foam created by aeration was automatically controlled with an aqueous suspension of silicone. Cultures were grown for 16 h to achieve maximum growth and toxin-antigen production (2). Preparation of crude toxin. All procedures for concentrating the supernatant fluid from fermenter cultures, as well as purification steps, were carried out at 4°C unless otherwise stated. The organisms were removed by centrifugation at 20,000 x g for 20 min, and the supernatant fluid was filter sterilized by passage through a prewashed 0.45-,Mm membrane filter (Millipore Corp., Bedford, Mass.). Merthiolate was added to the culture filtrate in a sufficient quantity to reach a bacteriostatic level (1:20,000). The filtrate was then concentrated 20-fold by pressure ultrafiltration in a model TCID ultrafiltration cell (Amicon Corp., Lexington, Mass.) using a UM10 Diaflo membrane. The effluents from all concentration steps were monitored for the presence of antigen by a passive hemagglutination test (2) before discarding.
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The concentrated retentate was then passed the toxin-antigen during the purification procedure, through an XM-100 Diaflo membrane to remove ma- samples were subjected to analytical disc electroterial of a molecular size greater than 100,000. The phoresis using acrylamide gel electrophoresis. PorXM-100 retentate was homogenized and subjected to tions containing 50 to 200 mg of protein from each ultracentrifugation at 100,000 x g for 2 h. The pellet step of purification, a small amount of sucrose, and was resuspended and washed twice in phosphate- tracking dye were applied to a 5.0% acrylamide gel buffered saline, pH 7.0, and the washings were slab. The gel was prepared in 0.1 M Tris-boratepooled with the XM-100 ultrafiltrate. The retentate ethylenediaminetetraacetate buffer, pH 8.5, and and washings were concentrated to a final volume of equilibrated by prerunning at 100 mA for 30 min. 30 ml on a UM-10 Diaflo membrane. This procedure The samples were subjected to electrophoresis at 50 is necessary to recover an additional 20 to 40% toxin- mA until the dye entered the gel, and the electroantigen "trapped" by the high-molecular-weight li- phoresis was then increased to 125 mA for the repopolysaccharide. mainder of the run. The gel slab was removed from Toxin purification. The concentrated preparation the cell and placed in 12% trichloroacetic acid for 2 was applied to a column (5 by 60 cm; Pharmacia Fine h. After fixation of the protein the gel was washed Chemicals, Inc., Piscataway, N.J.) packed with with tap water and placed in 0.25% Coomassie blue Bio-Gel agarose 0.5M (Bio-Rad Laboratories, Rich- for 5 to 6 h. The gel was then destained in an mond, Calif.) equilibrated with 5 mM sodium phos- electrophoretic destainer with methanol-water-glaphate buffer, pH 7.2, containing merthiolate cial acetic acid (5:5:1). The gels were stored in 5% (1:20,000). Elution with the equilibrating buffer at glycerine. a flow rate of 60 ml/h removed the larger-molecularsize lipopolysaccharide as well as the smallerRESULTS molecular-size medium constituents. These and The various steps involved in the purification subsequent column fractions were monitored continuously by absorbance at 280 nm with a Beckman of the cholera exoenterotoxin on a large-scale DB spectrophotometer (Beckman Instruments, Inc., basis are demonstrated by the flow diagram in Fullerton, Calif.) equipped with a flow cell. The Fig. 1. The steps involving membrane ultrafiltoxin-antigen fractions, recognized by the passive tration and ultracentrifugation deal with conhemagglutination assay, were pooled, adjusted to centrating 7 to 8 liters of culture filtrate to pH 6.5 with 5 mM monobasic sodium phosphate, approximately 30 ml. There is little purification and applied to a Bio-Gel AG50W cation-exchanger column (2.8 by 80 cm) equilibrated with 5 mM so- of the toxin-antigen during these steps, except dium phosphate buffer, pH 6.5. The toxin-antigen for the removal of the high-molecular-weight was eluted with 10 mM dibasic sodium phosphate material which is discarded in the washed peland concentrated to approximately 15 ml employ- let after ultracentrifugation. In addition, small ing a UM2 membrane. The concentrated toxin- amounts of low-molecular-weight material are antigen was subjected to preparative gel acrylamide discarded in the ultrafiltrates. electrophoresis using a Canalco Prep-Disc (Canal Gel filtration. The additlon of the concenIndustrial Corp., Rockville, Md.) equipped with a constant-voltage Buchler power supply (Buchler InCRUDE CULTURE FILTRATE struments Div., Nuclear-Chicago Corp., Fort Lee, 7.5 LITERS N.J.). Purification of the toxin-antigen was accomCONCENTRATED plished by passage through a 3.0-cm length of 4.0% UM-IO acrylamide stacking gel and through a 0.7-cm RETENTATE length of 7.0% acrylamide separating gel. The stackULTRAFILTRATE XM-100 DISCARD ing and separating gels were prepared in 0.1 M TrisRETENTATE 100,000 x g borate buffer, pH 8.5 and 9.5, respectively. After ULTRAFILTRATE PELLET WASH 2X PBS pH 74 polymerization of the gels with ammonium persul,| ~~~WASH INGS fate the apparatus was assembled, and the electrode WASHED PELLET chambers were filled with 0.06 M Tris-borate buffer, DISCARD CONCENTRATED pH 8.5. The column was then cooled to 4°C by circuUM-IO lating ice water through the cooling compartments. ULTRAFILTRATE RETENTATE A few crystals of sucrose were added to the sample to DISCARD BIO-GEL A-0.5 M permit layering on top of the stacking gel. A small r ANTIGEN FRACTIONS LIPOPOLYSACCHARIDE amount of tracking dye was added to monitor the BIO-GEL AG 50W LOW MW MATERIAL progress of the sample through the gel. Electropho| DISCARD EQUILIBRATING BUFFER EWTION ANTIGEN EWTED resis was started at 3.0 mA and continued until the DISCARD CONCENTRATED UM-2 sample entered the stacking gel. The current was then increased to 7.5 mA for an additional 5 h. RETENTATE ULTRAFILTRATE PREPARATIVE GEL DISCARD Elution was carried out with 0.1 M Tris-borate ELECTROPHORESIS buffer, pH 9.5. A peristaltic pump (Buchler InstruNON-ANTIGEN FRACTIONS ments) was used to maintain a flow rate of 40 ml/h. PURIFIED TOXIN ANTIGEN DISCARD The antigen content of the fractions was determined by the passive hemagglutination inhibition assay: FIG. 1. Flow diagram for cholera enterotoxin puAfter the removal of contaminating material from rification from TRY medium.
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from the toxin in this step, since the overall net charge of the two is so close at the pH employed for elutions. Preparative acrylamide electrophoresis. The pooled and concentrated fractions were then subjected to preparative gel acrylamide electrophoresis. A typical elution profile is shown in Fig. 3. The optical density of each fraction was plotted at both 280 and 260 nm to help locate the antigen. Titrations of the fractions were determined by the PHI assay and are plotted as micrograms of antigen per milliliter. Antigen elutes just after the tracking dye and unpolymerized acrylamide, which has a high 260-nm absorbance. Antigen elution begins slowly, reaches a plateau, and then emerges as a rather sharp peak. The fractions containing homogeneous toxin-antigen have absorbancy at 280 nm/absorbancy at 260 nm ratios approaching 2. A photograph of selected fractions and the starting material which were subjected to analytical gel electrophoresis can be seen in the upper left corner of Fig. 3. The antigen elution profile as well as the position of the protein bands on the analytical gel suggest the possibility of several species of toxin-antigen. This is supported by the fact that the antibody used in the PHI assay was made using material eluted in the sharp peak as the immunogen. The antigen fractions eluted first possess little or no biological activity (data not presented) but do react in the PHI assay. This may indicate that these species or subunits of toxin-antigen possess a high proportion of antibody reactive sites which are not biologically active. Previous experiments have shown that the heavy band seen in the concentrated starting material (Fig. 3) at the top of the analytical gel possesses no permeability factor activity but gives a reaction of identity with the purified toxin when reacted with monospecific antibody. In analogy to the data of Finkelstein and LoSpalluto (5), this material is toxoid. Elution of the naturally occurring toxoid from the preparative column can be accomplished by continuing electrophoresis for an additional 18 to 20 h. A more efficient means of obtaining purified toxoid is to reduce the percentage of acrylamide in the stacking gel from 4.0 to 3. 0%. Under these conditions the toxin peak and the tracking dye overlap, but the toxoid can be eluted in a 10- to 12-h period. Table 1 gives the percentage of recovery of micrograms of antigen and biological activity 4 12 20 28 36 44 52 60 68 76 for each step of the purification scheme. The FRACTION (5 ML) percentage of recovery is expressed as microculture fil- grams of antigen based on the PHI assay and as FIG. 2. Elution profile of TRY crude limit-of-bluing doses, with the activity of the trate from agarose 0.5M column. The toxin-antigen unconcentrated crude culture filtrate serving was determined by PHI.
trated toxin to the Bio-Gel agarose column (Fig. 1) is the first step in the removal of contaminating material. A typical elution profile from the agarose column can be seen in Fig. 2; the optical density at 280 nm and the micrograms of antigen per milliliter, determined by the PHI, are plotted for each 5-ml fraction. It is apparent that the material eluted in the column void volume contains very little antigen and represents molecules of 500,000 molecular size or greater (e.g., residual lipopolysaccharide). The toxin-antigen was eluted in the second peak and contains some colored material which apparently has a molecular size similar to the antigen. The dark-colored material eluted at the end of the column volume would have a molecular size of 5,000 to 10,000 and, for the most part, probably represents the low-molecular-weight medium constituents. Ion-exchange chromatography. The antigen-containing fractions from the agarose column were pooled and adjusted to pH 6.5 with 5 mM monobasic sodium phosphate. This material was added to the Bio-Gel AG50W cationexchanger column and eluted with 2 bed volumes of the equilibrating buffer or until the optical density reached zero. The antigen was eluted from the cation-exchanger with 10 mM dibasic sodium phosphate, pH 8.5. The ion-exchange step was carried out at room temperature, because the more concentrated fractions precipitate at lower temperatures at this pH. The pooled antigen fractions were concentrated on a UM-2 membrane to a total volume of 15 ml. An advantage of using the Bio-Gel cationexchanger is that it can be reconstituted in situ and can therefore be used for several purification cycles without having to repack the column. There is little separation of the toxoid
292
APPL. ENVIRON. MICROBIOL.
LEWIS, RICHARDSON, AND SHERIDAN
S20
W
2~~~~~~~~~~~~~~ / o~=~\\ N V500W
Nb'
.200
.~~~~~~~~~~~~~ .300
0 150
too1
.200
.100/100 2
+
(.
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10
12 1X
16
18
20
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24
FRArCTION (1 ML)
26 28
30
32
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381
40
e 4
FIG. 3. Elution profile of concentrated toxin-antigen from preparative gel acrylamide column. Antigen levels were determined by PHI. In upper left corner is a photograph of selected fractions and starting material (SM) which were subjected to analytical gel electrophoresis. TABLE 1. Summary of cholera toxin purification Material
CF'
Vol (ml)
Total protein (mg)
PHI 47 707
Antigen (,ug/ml)a Lf
RD
of Lb/,ug proteinb
Recovery_(%) PHI Lb
54 53 2.75 100 100 7,400 4,839.60 XM-100 495 712 690 3.07 100 100 4,515.00 UM-10 32.5 3,848.00 3.80 100 99 10,688 12,024 9,400 A-0.5M 670 385.92 396 427 26.12 76 75 430 AG50W 20 295.20 72 75 32.50 12,492 12,220 13,400 PA" 15 151.86 56 49.48 43 9,996 10,650 11,250 a Toxin antigen was determined by passive hemagglutination inhibition (PHI), limit of flocculation (Lf), and radial immunodiffusion (RD). Biological activity expressed as limit-of-bluing (Lb) doses. Crude culture filtrate. "Preparative acrylamide.
as 100%. Again, the first three steps of the purification scheme are concentration procedures with little or no loss of either antigen or biological activity. The 25% drop in both biological activity and antigen could not be detected in the 500,000-molecular-weight peak from the agarose column. This might be explained on the basis that the lipopolysaccharide is attaching to the antigen in such a way that the antigenic and/or biological sites are covered. This could in turn interfere with the detection of the
toxin-antigen in the various assays. The AG50W cation-exchange step appears to remove contaminating substances with a minimum loss of antigen or biological activity. The preparative acrylamide electrophoresis step results in an apparent loss of antigen, because only purified toxin is eluted. The loss of additional biological activity suggests some conversion of toxin to toxoid during fractionation by polyacrylamide gel electrophoresis. The final product had 56% of the total biological activity
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PURIFICATION OF CHOLERA ENTEROTOXIN
of the crude culture filtrate and was 18 times as toxic on a microgram of protein basis. DISCUSSION The specific activity of the purified toxin prepared by the technique described here ranges from 5 to 6 bluing doses per nanogram of toxinantigen, which is consistent with earlier reports from this laboratory (11, 12) as well as those from Finkelstein's laboratory (5, 6). In a more recent report, however, Finkelstein and LoSpalluto (7) state that crystallized cholera toxin has a specific activity of 20 bluing doses/ ng. Accepting this latter figure as "pure" would suggest that our final product is either heavily contaminated with other proteins or that the toxin as isolated is largely (two-thirds) biologically inert. There is no evidence of contaminating protein even on grossly overloaded analytical gel slabs. However, the diffuse multibanded appearance of some of the preparative gel fractions suggests that dissociation and reassociation of subunits may be occurring during electrophoresis. This could result in creation of inactive aggregates which would manifest themselves in lowered specific activity of the final product. The relatively long exposure to diluting buffers and charged conditions may also have an adverse effect on the toxin's biological activity without a concomitant reduction in immunochemical reactions. In summary, we have developed a method for the purification of choleragenic toxin from multiliter culture filtrates with 50 to 60% recovery of the starting biological activity. The purification scheme consists of concentration, gel filtration, ion-exchange chromatography, and preparative acrylamide gel electrophoresis as the final step. Although this procedure does not give greater yields than Finkelstein's technique, it does offer an alternative approach to procurement of reasonable amounts of purified toxin.
293
During the completion of this work, a chemically defined medium for production of cholera exoenterotoxin was formulated in our laboratory (1, 2). We have since adapted this defined medium to fermenter cultures and will present a purification scheme employing this chemically defined system in a future report. LITERATURE CITED 1. Callahan, L. T., III, and S. H. Richardson. 1973. Biochemistry of Vibrio cholerae virulence. III. Nutritional requirements for toxin production and the effects of pH on toxin elaboration in chemically defined media. Infect. Immun. 7:567-572. 2. Callahan, L. T., III, R. C. Ryder, and S. H. Richardson. 1971. Biochemistry of Vibrio cholerae virulence. II. Skin permeability factor/cholera enterotoxin production in a chemically defined medium. Infect. Immun. 4:611-618. 3. Coleman, W. H., J. Kaur, M. E. Iwert, G. J. Kasai, and W. Burrows. 1968. Cholera toxins: purification and preliminary characterization of ileal loop reactive type 2 toxin. J. Bacteriol. 96:1137-1143. 4. Craig, J. P. 1966. Preparation of the vascular permeability of Vibrio cholerae. J. Bacteriol. 92:793-795. 5. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Pathogenesis of experimental cholera-preparation and isolation of choleragen and choleragenoid. J. Exp. Med. 130:185-202. 6. Finkelstein, R. A., and J. J. LoSpalluto. 1970. Production of highly purified choleragen and choleragenoid. J. Infect. Dis. 121:S63-S72. 7. Finkelstein, R. A., and J. L. LoSpalluto. 1972. Crystalline cholera toxin and toxoid. Science 175:529-530. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Rappaport, R. S., B. A. Rubin, and H. Tint. 1974. Development of a purified cholera toxoid. I. Purification of toxin. Infect. Immun. 9:294-303. 10. Richardson, S. H. 1969. Factors influencing in vitro skin permeability factor production by Vibrio cholerae. J. Bacteriol. 100:27-34. 11. Richardson, S. H., D. G. Evans, and J. C. Feeley. 1970. Biochemistry of Vibrio cholerae virulence. I. Purification and biochemical properties of PF/cholera enterotoxin. Infect. Immun. 1:546-554. 12. Richardson, S. H., and K. A. Noftle. 1970. Purification and properties of permeability factor/cholera enterotoxin from complex and synthetic media. J. Infect. Dis. 121:S73-S79.
