Journal of Virological Methods, 3 (1982) 349- 354

Elsevier Biomedical Press

349

LARGE-SCALE ISOLATION OF THE TWO MAJOR POLYPEPTIDES OF THE

HEPATITIS B SURFACE ANTIGEN (HBsAg) BY GEL FILTRATION IN 6 M

GUANIDINIUM CHLORIDE

MICHEL HUBERT, FRANCIS BARIN and ALAIN GOUDEAU

Institut de Virologie, Facultt? de Wdicine et de Pharmucie, 2 his, Boulevard TonnelIP, 3 7000 Tours,

France

(Accepted 4 September 1981)

Purified hepatitis B surface antigen (HBsAg) of subtype ay was solubilized in guanidinium chloride

and submitted to chormatography on Sepharose 4B in the presence of guanidinium chloride. The

polypeptides Pl (MI = 24,000) and P2 (Mr = 29,000) were eluted in the same fraction with a minor

contaminant (M, = 40,000). Large amounts of these two polypeptides were obtained in a single step.

This technique which constitutes a method for large-scale purification of the Pl and P2 polypeptides

should permit more complete characterization of the Pl and P2 polypeptides and of their antigenic

determinants.

hepatitis B HBsAg polypeptides large-scale purification

INTRODUCTION

Over the last five years, the safety, potency, and efficacy of a vaccine against hepatitis

B were shown to be very satisfactory in patients and staff of haemodialysis units of the

Loire Valley (France) (Maupas et al., 1976, 1978; Doniach et al., 1979). Although this

vaccine contains only the purified formalin-inactivated surface antigen (HBsAg) of the

hepatitis B virus (HBV), some arguments on an immunological basis were advanced

against the use of HBsAg of human origin as a vaccine (Zuckerman, 1976). Therefore,

polypeptides isolated from HBsAg were considered for the preparation of hepatitis B

vaccines excluding host-derived substances (Dreesman et al., 1975).

Hepatitis B surface antigen is known to contain two major polypeptide species of

molecular weight 22,000-26,000 (Pl) and 28,000-30,000 (P2), irrespective of the

origin of HBsAg, i.e. human serum (Germ et al., 1975; Peterson et al., 1977; Cabral et al.,

1978) or human hepatoma cell line (Monjardino and Crawford, 1979; Skelly et al.,

1979b). However, additional major components are frequently found in the molecular

weight range of 35,000-125,000, according to the method of purification and the

source of material used. Among these additional polypeptides, one has been identified

as albumin (Skelly et al., 1979a; Shih et al., 1980).

0166-0934/82/0000-0000/$02.?5 OElsevier Biomedical Press

The two polypeptides (Pl and P2) have a very similar amino acid composition (Peter-

son et al., 1977; Shih and Gerin, 1977b) and identical amino acid sequences of the amino-

and carboxy-terminals (Peterson et al., 1978). It has been suggested that P2 differs from

Pl by glycosylation only (Peterson et al., 1977). The structural gene coding for these

polypeptides has been identifed on the viral genome (Charnay et al., 1979; Valenzuela et

al., 1979). This gene corresponds to a theoretical polypeptide with a molecular weight

of 25,400. To date, HBsAg polypeptides have been purified by preparative SDS-poly-

acrylamide gel electrophoresis (SDS-PAGE) but only in small amounts, or by affinity

chromatography of HBsAg solubilized with Triton X-100 (Skelly et al., 1979a). In the

present study, a method for large-scale purification of the polypeptides from HBsAg is

described using gel filtration in the presence of guanidinium chloride.

MATERIALS AND METHODS

Source of purified HBsAg

The antigen was purified from HBsAg-positive sera, subtype ay, from three healthy

subjects. The method of purification of HBsAg has been described previously (Barin et

al., 1978). Briefly, the four steps of the purification were successively: selective adsorption--

desorption on colloidal silicate (Aerosol R 380 from Degussa, Frankfurt, F.R.G.), pre-

cipitations by polyethylene glycol 6000, gel filtration, and finally isopycnic banding in

CSCI.

Fractionation of HBsAg polypeptides by gel filtration in 6 M guanidinium chloride (GuHcI)

HBsAg polypeptides were isolated according to the technique described by Green and

Bolognesi (1974). Lyophilized HBsAg was solubilized in 1 .O ml of 8 M GuHCl (Sigma

Chemical Comp., St. Louis, MO), 0.15 ml of mercaptoethanol and 10 mg of EDTA

(trisodium salt) for 4 h at 50°C.

This sample was layered on a 1.6 X 90 cm column of Sepharose 4B (Pharmacia Fine

Chemicals), equilibrated and eluted with 6 M GuHCl, 0.02 M mercaptoethanol buffered

with 0.05 M sodium acetate, pH 5.0. One millilitre fractions were collected, with a flow

rate of 4 ml/h, while monitoring absorbance at 280 nm with a LKB 2 138 Uvicord S spec-

trophotometer (LKB Produkter AB, Bromma, Sweden). Guanidinium chloride was

removed from the samples by dialysis against two changes of 2 mM EDTA containing

10 mM mercaptoethanol, followed by dialysis against two changes of 5 mM mercapto-

ethanol.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Purified HBsAg and polypeptide fractions were solubilized by incubation at lOO”C,

351

for 2 min, in 0.01 M sodium phosphate buffer, pH 7.2, containing 1% (w/v) SDS, 1%

(v/v) mercaptoethanol, 10% (v/v) glycerol, and 0.02% (w/v) bromophenol blue. Then,

samples were analyzed by SDS-PAGE according to the technique of Weber and Osborn

(1969). Polymerized gels contained 10% (w/v) acrylamide, 0.27% (w/v) methylene bis-

acrylamide, 0.075% (w/v) ammonium persulfate, 0.15% (v/v)N, N, N’, N’-tetramethylene

diamine and 0.1% (w/v) SDS in 0.1 M sodium phosphate buffer, pH 7.2. The electro-

phoretic buffer was composed of 0.1 M sodium phosphate buffer, pH 7.2, containing

0.1 %(w/v) SDS and 0.1 %(v/v) mercaptoethanol.

Molecular weights of the polypeptides were estimated using the Pharmacia low mole-

cular weight electrophoresis calibration kit (Pharmacia Fine Chemicals, Uppsala, Sweden),

with a semilogarithmic plot of the protein molecular weight versus their relative migration.

Samples containing 30 - 150 pg of protein were layered onto each 6 X 85 mm gel and

submitted to electrophoresis at 8 mA/gel for 3.5 h. After staining with 0.25% (w/v)

Coomassie blue R 250 in a mixture of methanol and acetic acid as described by Weber

and Osborn (1969), the gels were scanned at 5 80 nm using the Super Cellomatic spectro-

photometer (SEBIA, Issy les Moulineaux, France).

RESULTS

SDS-PAGE analysis of purified HBsAg preparation resulted in four major polypep-

tides with molecular weights of 50,000, 38,000, 29,000, 24,000, and additional minor

components, some of which having a low molecular weight (< 20,000).

After complete solubilization in GuHCl, mercaptoethanol and EDTA, 22 mg of puri-

fied HBsAg were chromatographed on Sepharose 4B equilibrated with 6 M GuHCl, 0.02 M

mercaptoethanol (pH 5 .O). The elution profile showed five fairly sharp major peaks (Fig.

1). The fractions were combined in seven pools (I-VII) which were dialysed as described

above, lyophilized and analysed by SDS-PAGE. The polypeptides were eluted by de-

o.21’a = : = ’

;:1;:-: 150 175 :

Fraction number

m

Fig. 1. Sepharose 4B chromatography (1.6 X 90 cm) of purified HBsAg solubilized by guanidinium

chloride (GuHCl). Elution was performed with 6 M GuHCl, 0.02 M mercaptoethanol buffered with

0.05 M sodium acetate, pH 5.0, at a flow rate of 4 ml/h. One milliliter fractions were collected.

352

creasing order of molecular weight (Fig. 2). The two polypeptides PI and P2 with mole-

cular weights of 24,000 and 29,000 were located in pool V along with a minor contami-

nant with a molecular weight of 40,000 giving a total protein content of 3 mg (Fig. 3).

DISCUSSION

Purified HBsAg of subtype ay consisted essentially of four polypeptides with mole-

cular weights of 24,000, 29,000, 38,000 and 50,000, when analysed by SDS-PAGE.

These results are similar to those obtained by different authors who describe two major

polypeptides Pl (M, = 22,000-26,000) and P2 (M, = 28,000-30,000) (Gerin et al.,

197.5; Peterson et al., 1977; Cabral et al., 1978; Monjardino and Crawford, 1979: Skelly

et al., 1979b). However, in contrast with other authors who described a third major

polypeptide with a molecular weight of 64,000-72,000 (Shih and Gerin, 1977a; Skelly

et al., 1979a), this study revealed the presence of an important polypeptide with a mole-

cular weight of 50,000.

Considering that the gene S coding for both polypeptides Pl and P2 has been localized

on the HBV genome (Charnay et al., 1979; Valenzuela et al., 1979), the study of these

two polypeptides of specific viral information is of great interest. For this reason, poly-

peptides Pl and P2 have been obtained on a large-scale basis from purified HBsAg. The

preparation of Pl and P2 was achieved by gel filtration in the presence of guanidinium

chloride. On the basis of stain intensity obtained after SDS-PAGE, pool V contained

approximately 93% of P2, 5% of PI and 2% of a 40,000 mol. wt. polypeptide.

(BJ

(CJ

(DI

(EJ

(F)

94 ooo-

67 OOO-

43 ooo- -50 000

30 ooo-

/ -24 000

20 loo-

14 400-

0 II III IV V HBsAg

I P2)

(PI)

Fig. 2. SDS-PAGE of purified HBsAg and of polypeptides derived from pooled gel filtration fractions

illustrated in Fig. 1. Standards used included: A) phosphorylase b, B) albumin, C) ovalbumin, D) car-

bonic anhydrase, E) trypsin inhibitor, F) a-lactalbumin. Gels were stained for protein with Coomassie

blue.

353

Fig. 3. Densitometric scans of the gels corresponding to the purified HBsAg and pool V shown in Fig. 2.

Polypeptides Pl and P2 were eluted simultaneously in pool V. The isolation of Pl and

P2, individually, was not possible under these conditions. It could be realized with a

longer column or by recycling the interesting fraction. The difficulty of separating these

two polypeptides by such a technique seems to confirm the view of Peterson et al. (1977)

that the real difference in the molecular weights of Pl and P2 is not marked. This would

be due to the overestimate of glycoprotein molecular weights by SDS-PAGE (Weber et

al., 1972).

354

The purification technique described in this report allows rapid and easy isolation of

large amounts of HBsAg-derived polypeptides. Using only one step, 22 mg of solubilized

HBsAg were analysed by chromatography. But it should be noted that amounts of

solubilized HBsAg up to 60 mg could be layered on such a column. By this technique,

the isolation of large quantities of Pl and P2, which are the major polypeptides of HBsAg

excluding host-derived material, would allow a more complete characterization of these

polypeptides and study of their antigenic determinants. The use of such haptenic deter-

minants would constitute a new approach towards a second generation hepatitis B vaccine

(Zuckerman et al., 1981).

ACKNOWLEDGEMENTS

This work was supported by contract No. PBP/p88 bis from the Delegation a la

Recherche Scientifique et Technique (DGRST), France.

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