JOURNAL oP BACTERIOLOGY, Sept. 1967, p. 580-585 Vol. 94, No. 3Copyright @ 1967 American Society for Microbiology Printed In U.S.A.

Tetanus Toxin and Antigenic DerivativesI. Purification of the Biologically Active Monomer

SAMUEL G. MURPHY AND KENT D. MILLERDivision of Laboratories and Research, New York State Department of Health, and Department of

Microbiology, Albany Medical College, Albany, New York 12201

Received for publication 17 June 1967

A procedure for the isolation of pure tetanus toxin in a lethal monomeric formwas developed based on the extraction of whole cells and chromatographic tech-niques. A crude extract of toxin was obtained by hypertonic extraction of cellsfrom a 72-hr culture of Clostridium tetani Massachusetts strain. The extract wasprecipitated with ammonium sulfate and further purified by sequential use of ion-exchange chromatography and gel filtration. The degree of purification obtainedby the fractionation procedures was monitored by polyacrylamide gel electro-phoresis. The pure toxin has an average specific activity of 150 x 106 mouse MLDper mg ofN and 3,000 Lf per mg of N. Immunological purity was demonstrated bya single line on both immunoelectrophoresis and agar double diffusion. One bandwas obtained on polyacrylamide electrophoresis, as was a single symmetrical peakin the ultracentrifuge and on Sephadex G-100 chromatography. The pure proteinhas an absorbancy ratio (280/260 mi,) of 2.1 in phosphate buffer (pH 7.5).

Tetanospasmin, the neurotoxin produced byClostridium tetani, has been subjected to manyfractionation procedures. Prior to 1959, precipi-tation techniques were the most widely employedprocedures. [Reports on their use were reviewedby Turpin and Raynaud (18)]. Recently, severalchromatographic purifications have been re-ported. For these procedures, either ion-exchangechromatography (1, 8, 15, 17) or gel filtration (7,15) has been used. None of the preparations ob-tained by these methods has been shown to be asingle component with respect to both im-munological and chemical criteria of purity.

In an attempt to produce an immunogen freefrom other protein contaminants, culture fil-trates from autolyzed cells were precipitated orultrafiltered prior to purification on a technical-scale Sephadex G-100 column (20 x 120 cm).These procedures were abandoned as inoperable.It was necessary to explore other methods thatwould allow smaller working volumes and theapplication of additional parameters of separa-tion to obtain pure tetanus toxin.

MATERIALS AND METHODS

Chromatographic materials. Microgranular di-ethylaminolethyl (DEAE) cellulose, Whatman DE-52,obtained from H. Reeve Angel and Co., Inc., Clifton,N.J., was cycled in 0.5 N HCl and 0.5 N NaOH priorto equilibration in 0.05 M tris(hydroxymethyl)amino-methane (Tris) chloride buffer (pH 7.5). SephadexG-50, fine, and Sephadex G-100 were obtained fromPharmacia Fine Chemicals, Inc., Piscataway, N.J.

The Sephadex G-50 was used as supplied. SephadexG-100 was sieved on a Tyler Portable Sieve Shaker(Fisher Scientific Co.. Pittsburgh, Pa.) fitted withU.S. Standard Sieve Series, 63- and 88-, screens. The63- to 88-, particles were used in both preparative andanalytical Sephadex G-100 columns.

Preparation of crude cell extract. C. tetani Massa-chusetts strain was obtained from W. C. Latham,Massachusetts Department of Public Health, Instituteof Laboratories, Boston, Mass. A modified Muellermedium (6), 8 liters in 9-liter bottles, was inoculatedand incubated at 33 C for 72 hr. In preliminary experi-ments 30-liter cultures were employed; in later ex-periments, 100-liter lots were used. Bacteria wereharvested by continous celntrifugation at room temper-ature in a DeLaval Gyrotester (The DeLavel Separa-tor Co., Poughkeepsie, N.Y.). The cell paste was re-moved after each centrifugation of 50 liters of medium.Figure 1 is a flow diagram of the purification proce-dure.The cell paste was resuspended in an extraction

fluid consisting of 1.0 M NaCl and 0.1 M Na citrateaccording to the elegant method of Raynaud (13).The cells were extracted for 5 days at 4 C, and theresulting extract (CEX) was separated from cell debrisby centrifugation at 10,000 X g for 20 min at -5 Cin a Sorvall RC-2 centrifuge with a GSA head.

The cell extract (CEX) was precipitated by theaddition of 470 g of solid (NH4)2SO4 per liter of CEX.The pH was adjusted to 5.0 with HCl. The precipitatewas allowed to settle overnight at 4 C. It was collectedby centrifugation in a Sorvall RC-2 as described aboveand dissolved in 0.05 M Tris chloride buffer (pH 7.5).This concentrated CEX was desalted on SephadexG-50 (7.5 X 90 cm) in 0.025 M Tris chloride buffer(pH 7.5). All chromatography was performed at 4 C.

580

on May 24, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

TETANUS TOXIN AND ANTIGENIC DERIVATIVES. I

72 hr Culture C. tetaniCentrifuge

Supernatont Cells

resuspend in EXTRACTIONfluid; O.l culture volume

Extract 5 days -

centrifuge

C E X Cell debris

Supernatant

470g(NH4)2S04/ literCCe nt ri fuge

ppt -

red issolve in 0.09Extraction fluid volume

Desa It on Sephadex G- 50

DE AE Cellulose

pool tox in peak -

ppt 300g( NH4)2S04 / iter

redissolveppt inminimum vol.

Sephadex GIOOFIG. 1. Flow diagram for the purification of tetanus

toxin. This procedure was employed on lots of 30 to200 liters without modification.

Chromatography of CEX. The desalted CEX wasadsorbed on a DEAE cellulose column previouslywashed until the effluent was equal to the influentbuffer with respect to pH and conductivity. The toxinwas eluted with a linear gradient of 0.05 M Trischloride (pH 7.5) to 0.05 M Tris buffer (pH 7.5), 0.1M NaCl.

Pooled selected fractions from the DEAE cellulosecolumn were precipitated by adding solid (NH4)2SO4to 0.5 saturation. The precipitate was redissolved in0.1 M phosphate buffer (pH 7.5) and chromatographedon Sephadex G-100, 63 to 88 At (5.0 X 235 cm),equilibrated in 0.1 M phosphate buffer (pH 7.5).

All columns were eluted at a constant flow rate. Amodified Micro bilateral roller pump (The HolterCo., Bridgeport, Pa.) was used as a metering pumpto slow the flow rate.

Analytic procedures. Spectrophotometric readingsof column effluents at 260 and 280 m,u were made ona Gilford model 1200 photometer attached to a Beck-man DU spectrophotometer. Conductivity of theDEAE cellulose elution was measured with a conduc-tivity bridge (model RC 16B2; Industrial Instrument,Cedar Grove, N.J.).

Polyacrylamide gel electrophoresis was performedby a modification of the Hjerten procedure (3). Thebasic buffer system was Tris phosphate (pH 8.6) witha final molarity of 0.07 M. The samples were appliedin 20% sucrose overlaid on the gel. A loading cur-rent of 2 ma/tube was applied for 15 min, and thecurrent was then increased to 5 ma/tube for 1 hr.

Mouse minimal lethal dose (MLD) was deter-mined by intramuscular injection of 0.5 ml of the ap-propriate dilution into the thigh of each of five 16- to20-g white Swiss mice (11).

Flocculating parameters, Lf and Kf, were deter-mined by the method of Dean and Webb (2). In alltests, the concentration of the antibody solution was50 Lf per ml as standardized against reference tetanus

(n0

II

E

TUBE NUMBER

FIG. 2. Chromatography of CEX on DEAE cellu-lose column (2.5 X 20.0 cm). CEX (350 ml) was ad-sorbed, and then eluted with a 1.0- liter linear gradient.Tubes 40 to 80 contained 90% of the total toxin. Frac-tion volume, 7.0 ml. Solid line, absorbance at 280 m,u;dotted line, absorbance at 260 m,; X, conductance.

cnz

0

-j

a-

0

30 34 38 42 46 50

TUBE NUMBER

FIG. 3. Gel filtration of concentrated CEX andpure toxin on Sephadex G-100, 63 to 88 ,A (1.8 X 170cm). Sample volume, 5.0 ml. Fraction volume, 4.5 ml.Flow rate, 3 ml per cm2 per hr. CEX: heavy solid line,absorbance at 280 miA; dashed line, absorbance at 260m,u. Pure toxin: light solid line, absorbance at 280 mM;dotted line, absorbance at 260 m,.

VOL. 94, 1967 581

s

on May 24, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

MURPHY AND MILLER

antitoxin furnished by the State Serum Institute ofCopenhagen. The final volume in all tubes was ad-justed to 1.5 ml with saline. The tubes were incubatedat 50 C, and the first flocculation was chosen as theLf. Immunological purity was assayed by agar doublediffusion (10) and by immunoelectrophoresis (16).

-30

z 1 )

ui ~~~~~~~~~~02.0

<

Sedimentation velocity was determined with a

Spinco Model E ultracentriguge. Kjeldahl nitrogendeterminations were performed according to methodsdescribed by Kabat (4).

RESULTS

Preliminary experiments were carried out on

more than twenty 30-liter preparations with onlyslight variations in yield and purity noted. Chro-matography of the desalted CEX on DEAEcellulose is illustrated in Fig. 2. Tetanus toxin iseluted in this chromatogram at a conductance ofapproximately 2.5 mmhos, 4C. In Fig. 3, thechromatography of purified toxin on SephadexG-100 is compared with that of the crude CEX.The point of tetanus toxin elution is the onlypoint where the absorbance at 280 m,i exceedsthat at 260 m,u in the elution of CEX.

40 80 '20 160 200 240

TUBE NUMBER

FIG. 4. Preparative scale chromatography of CEXfrom 100-liter culture on DEAE cellulose (5 X 15 cm).CEX (1,000 ml) was adsorbed and eluted with a 4-literlinear gradient. Fraction volume, 12.0 ml. Solid line,absorbance at 280 m,u; dotted line, absorbance at 260m,u; X, conductance.

..-..4g

en-zLLI0

-J

(-

0.

0 20 40 60TUBE NUMBER

FIG. 5. Final purification of toxin by gel filtrationon Sephadex G-100, 63 to 88 ,u (5.0 X 235.0 cm). Flowrate, 3 mlper cm2 per hr. Fraction volume, 12 ml. Solidline, absorbance at 280 m,; dotted line, absorbance at260 m,u.

FIG. 6. Polyacrylamide gel electrophoresis offrac-tions from (reading from left to right) the sample andtubes 56, 70, 80, 100, and 160 of the DEAE celluloseelution (Fig. 4).

FIG. 7. Polyacrylamide gel electrophoresis of frac-tions from (readingfrom left to right) tubes 10, 31, 36,46, and 56 of the Sephadex G-100 elution (Fig. 5).

582 J. BACTERIOL.

400.mio....I......

.W 40w*

on May 24, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

TETANUS TOXIN AND ANTIGENIC DERIVATIVES. I

Since the basic chromatographic pattern forDEAE cellulose was established in preliminaryexperiments, the elution of the 100-liter prepara-tions was terminated at conductance of 5.0mmhos, 4C. In a typical 100-liter preparation(Fig. 4), fractions 80 to 160 were pooled, pre-cipitated with solid (NH4)2SO4, and redissolvedin buffer to a volume of 50 ml. This solution con-tained approximately 825 optical density (OD)units (at 280 m,). It was applied to the SephadexG-100 column (Fig. 5). Polyacrylamide electro-phoresis of fractions taken at intervals during theDEAE cellulose and Sephadex G-100 chromato-grams is shown in Fig. 6 and 7.

Fractions 33 to 49 from the Sephadex G-100column were pooled and precipitated with solid(NH4)'SO4 at 0.5 saturation. The precipitate wasredissolved in phosphate buffer (ionic strength0.2, pH 6.0) to a protein concentration of 1.5%and desalted in the same buffer on Sephadex G-50.Pure toxin did not denature and precipitate out ofsolution when stored at a protein concentrationof 1% in the phosphate buffer.

Immunological purity was demonstrated byboth immunoelectrophoresis and agar doublediffusion. Figure 8 demonstrates that the pureprotein yields one line of precipitation. No addi-tional lines were obtained for the pure toxinwhen the concentration of antibody or toxin wasvaried between 50 and 1,000 Lf and the constantcomponent was 200 Lf.The pure protein has an absorbancy ratio

(280/260 m,I) of 2.1 at 1.0 OD at 280 mA, withBeers law followed between 1.5 and 0.3 OD at280 m,A. Figure 9 is a plot of OD versus N used

(I)zL J

1.0

U oH 0

0~0

0 1.0 2.0mg Nx 10'

FIG. 9. Standard curve of OD versus nitrogen forpure tetanus toxin.

to convert MLD and Lf specific activity valuesfrom OD at 280 m,s to milligrams of N.The ultracentrifuge patterns in Fig. 10 demon-

strate a single component with an observed Svalue of 6.4. Within the limits of all assays em-ployed, the final product described above is apure, homogeneous protein.The yield and properties of this separation pro-

cedure are demonstrated in Table 1.

DISCUSSIONLarge volumes of tetanus culture filtrates have

been precipitated in this laboratory with methanoland ammonium sulfate but, because of the diffi-

FIG. 8. Agar double-diffusioni pattern of CEX (1)

and pure toxin (2), 500 Lf/ml. Horse anticrude tetanusantibody was employed at a concentration of500 Lf/ml.

FIG. 10. Ultracentrifuge pattern of tetanus toxin at16 min (left) and 48 min (right) after reaching speedof59,780 rev/min. The protein concentration was 0.5%in a pH 6.0 phosphate buffer, ,u = 0.2. Protein concen-trations of I and 1.5% demonstrated a single peak withthe same S value.

583VOL. 94, 1967

on May 24, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

MURPHY AND MILLER

TABLE 1. Yield and properties of tetanus toxin from a 100-liter culture

OD/mlPurification step Vol Lf/OD280 MLD/Lf Lf (%recovery)

260 my 280 ns|

72-hr culture 100 litersCEX 10 liters 5.0 3.50 14.3 100Concentrated CEX 900 ml 24.0 17.80 29.2 30,000 90Desalted CEX 1,000 ml 16.0 11.70 34.0 80DEAE cellulose tubes 80-160 800 ml 0.50 1.05 360.0 60Sephadex G-100 tubes 33-49 195 ml 1.80 3.75 360.0 50,000 53

culty in collecting the precipitate comparedwith harvesting whole bacteria, the extractionprocess appears far superior. The advantages ofusing a cell extract rather than a culture filtratewere described by Raynaud and co-workers (13,18). The data confirm that extraction of cellsfacilitates the purification of tetanus toxin inthree important ways: (i) culture media and extra-cellular products are removed from the bacteriaduring centrifugation; (ii) fewer antigens arepresent in the CEX than in culture filtrates; and(iii) the 10-fold reduction in volume facilitatesprocessing. These procedures have been used toprocess 200-liter lots, and larger culture volumescould probably be handled with the same equip-ment.

Gel filtration is superior to dialysis for desalt-ing and exchanging buffers prior to chromatog-raphy. The denaturation of toxin is minimized,presumably due to the shorter exposure to sur-faces required in gel filtration to obtain equiva-lent desalting.The product from the DEAE cellulose column

is approximately 95% pure. Most of the remain-ing contaminating material is removed by thesucceeding chromatography on Sephadex G-100.However, both the leading and trailing edges fromthe Sephadex chromatogram are frequently stillcontaminated and must be discarded or reproc-essed. For this reason, the leading edge, center,and trailing edge of all toxin peaks must bemonitored before fractions are pooled. The poly-acrylamide electrophoresis is a particularly sensi-tive tool for general monitoring during toxinseparations. Its superiority in this case is exem-plified by comparison of the desalted CEX asanalyzed by polyacrylamide electrophoresis (Fig.5) and by agar double diffusion (Fig. 7).Another important test for the presence of trace

contaminants, especially nucleic acids or de-natured products is deviation of the 280/260 m,uratio from the 2.1 range. At protein concentra-tions greater than 2 mg/ml there is a decrease inratio to approximately 2.0, presumably owing tolight scattering. However, material with a ratio

less than 2.0 has been found impure when ex-amined by polyacrylamide electrophoresis.Gel filtration, excellent for the final purification

of toxin, did not yield sufficiently pure materialwhen used as the only chromatographic step,even on longer columns than reported here. Thefirst half of the peak always contained nucleicacids and other proteins, whereas the last partof the peak was contaminated with the peptidaseoriginally described by Miller et al. (9). Thesedata do not confirm the work of Salenstedt andTirunarayanan (15), who stated that gel filtrationyielded a greater purification than did ion-ex-change chromatography.The properties of tetanus toxin isolated by our

procedure are similar to those reported by otherinvestigators (1, 18). It is difficult to compare Lfvalues due to the different reference standardsemployed by other investigators. In addition, thequantitation of the test is limited by replicatedilutions necessary to obtain readable floccula-tions. The MLD assay is even less precise becauseof the influence of mouse weights and environ-ment. We have not found the MLD to vary bygreater than 20% from 150 X 106 MLD per mgof N for freshly isolated matkrial. With storagetime, there is a reduction in MLD but no ob-served physical change in the molecule. It seemspossible that the great differences in MLD valuesobserved by Turpin and Raynaud (18) and Daw-son and Mauritzen (1) were differences in thedegree of denaturation during the isolation pro-cedure.The protein molecule itself can become de-

natured and precipitate (1), as we have also ob-served. This denaturation is especially true whendilute toxin is in contact with glass, cellulose, orcollodion membrane. The latter fact may havecaused some of the minor peaks in the ultra-centrifuge that were observed by Dawson andMauritzen (1).

Pillemer (12) and Largier (5) were the firstinvestigators to report values for the sedimenta-tion coefficient of the toxin. They reported valuesof 4.5 and 3.9S, respectively, for the biologically

584 J. BACTERIOL.

on May 24, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

TETANUS TOXIN AND ANTIGENIC DERIVATIVES. I

active component. In addition, they reported Svalues of 7.0 and 7.6 for an atoxic dimer. Thedimer was reported as formed spontaneouslywithin a few days at 4 C, but this has not beenconfirmed. Our values for the toxin, observedS = 6.4, agree with those obtained by Raynaudet al. (14) and Dawson and Mauritzen (1), evenon material stored for 3 weeks at 4 C.

Because of the consistency of the isolated ma-terial in both the biological and chemical assays,we propose that the material is the biologicallyactive monomer. We have been unable to isolatea smaller or larger lethal fraction from threedifferent starting materials: ultrafiltered or pre-cipitated culture filtrates and CEX.The pure tetanus toxin is obtained in good yield

and in adequate quantity to permit investigationof its use as a reagent-grade immunogen. Its useas an immunogen and its physical characteriza-tion are under investigation and will be reportedlater.

ACKNOWLEDGMENTmThis investigation was supported by research grant

HE-09902 and general research support grant 5-01-FR-5559-03 from the National Institutes of Health.The technical assistance of Patricia Apple and

Arthur Marin is gratefully acknowledged.

LITERAruRE CrrED1. DAWSON, D. J., AND C. M. MAURITZEN. 1967.

Studies on tetanus toxin and toxoid. I. Isola-tion of tetanus toxin using DEAE-cellulose.Australian J. Biol. Sci. 20:253-263.

2. DEAN, H. R., AND R. A. WEBB. 1926. The influ-ence of optimal proportions of antigen andantibody in the serum precipitation reaction. J.Pathol. Bacteriol. 29:473-492.

3. HJERTEN, S., S. JERSThT, AND A. T1sELIs. 1965.Some aspects of the use of "continous" and"discontinuous" buffer systems in polyacryl-amide gel electrophoresis. Anal. Biochem. 11:219-223.

4. KABAT, E. A. 1961. Experimental immunochem-istry, 2nd ed. Charles C Thomas, Publisher,Springfield, Ill.

5. LARGIER, J. F., AND F. J. JOUBERT. 1956. Investi-gation of the physical properties of tetanustoxoids. Biochim. Biophys. Acta 20:407-408.

6. LATHAM, W. C., D. F. BENT, AND L. LEVINE. 1962.Tetanus toxin production in the absence ofprotein. Appl. Microbiol. 10:146-152.

7. LATHAM, W. C., C. P. JENNESS, R. J. K. TIMPERI,C. B. H. MICHELSEN, E. M. ZIPILIVAN, G.EDSALL, AND H. L. LEY, JR. 1965. Purificationand characterization of tetanus toxoid andtoxin. I. Fractionation of tetanus toxoid by gelfiltration. J. Immunol. 95:487-493.

8. MARR, A. G. M., AND R. W. PATTERSON. 1960.The chromatographic isolation of tetanus toxinon diethylaminoethyl cellulose columns. Aus-tralian J. Sci. 23:131-133.

9. MILLER, P. A., C. T. GRAY, AND M. D. EATON.1960. Formation and action of a peptidasewhich hydrolyzes histidine peptides required intetanus toxin synthesis. J. Bacteriol. 79:95-102.

10. OUCHTERLONY, 0. 1948. In vitro method fortesting the toxin-producing capacity of diph-theria bacteria. Acta Pathol. Microbiol. Scand.25:186-191.

11. PILLEMER, L. 1946. The immunochemistry oftoxins and toxoids. I. The solubility and pre-cipitation of tetanal toxin and toxoid in meth-anol-water mixtures under controlled con-ditions of pH, ionic strength and temperature.J. Immunol. 53:237-250.

12. PILLEMER, L., R. G. WrIrLER, J. I. BURRELL, ANDD. B. GROSSBERG. 1948. The immunochemistryof toxins and toxoids. VI. The crystallizationand characterization of tetanal toxin. J. Exptl.Med. 88:205-221.

13. RAYNAUD, M. 1951. Extraction de la toxinetetanique 'a partir des corps microbiens. Ann.Inst. Pasteur 80:356-377.

14. RAYNAUD, M., A. TURPIN, AND B. BIzzIM. 1960.Existence de la toxine tetanique sous plusieursetats d'agr6gation. Ann. Inst. Pasteur 99:167-172.

15. SALENSTEDT, C. R., AND M. 0. TIRUNARAYANAN.1966. Purification of tetanus toxin with the aidof Sephadex gels. Z. Immunitaetsforsch. 130:190-196.

16. SCHEIDEGGER, J. J. 1955. Une micro-methode del'immuno-electrophorese. Intern. Arch. AllergyAppl. Immunol. 7:103-110.

17. SHEFF, M. F., M. B. PERRY, AND S. I. ZACKS. 1965.Studies on tetanus toxin. II. The preparationof purified tetanus toxin and its fluorescentlabelling. Biochim. Biophys. Acta 100:215-221.

18. TURPIN, A., AND M. RAYNAUD. 1959. La toxinet6tanique. Ann. Inst. Pasteur 97:718-732.

VOL. 94, 1967 585

on May 24, 2018 by guest

http://jb.asm.org/

Dow

nloaded from