Microbiol. Immunol. Vol. 36 (4), 419-423, 1992

A Simple Purification Method of VibriocholeraeNon-O1 Hemagglutinin/Protease by Immunoaffinity Column Chromatography Using a Monoclonal Antibody Atsuko

NAKA,* Takeshi

HONDA, and Toshio

MIWATANI

ResearchInstitute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan (Accepted for publication, December 27, 1991)

Abstract A new simple purification method (I) for Vibriocholeraenon-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Olichterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.

Vibrio choleraenon-O1 (NAG vibrio), which is similar to V. cholerae O1, but is not agglutinated by V. cholerae O1 antiserum, is an important cause of human diarrheal diseases. Gastroenteritis associated with V. choleraenon-O1 shows various clinical symptom (2, 12), such as abdominal pain, mucous and bloody diarrhea as well as watery diarrhea similar to cholera due to V. cholerae O1. These various symptoms

may

be explained

by the production

of a variety

of toxins

by V. cholerae

non-O1, including cholera toxin (CT), CT-like toxin (14), NAG-ST (heat-stable enterotoxin) (1), E 1 Tor-like hemolysin (15), TDH (thermostable direct hemolysin) like hemolysin (16), as well as hemagglutinin/protease (NAG-Ha/P) (4, 7). A series of observations (3, 5, 6, 13) indicated that hemagglutinin/protease (Vc-HA/P) of V. cholerae O1 may be involved in the pathogenesis of cholera, by mediating the intestinal attachment of the organisms, activating cholera toxin, and so on. NAG-HA/P was recently purified by us (10) using combinations of four chromatography columns and partial characterization suggested identity between NAG-HA/P and Vc-HA/P. However, as the purification of NAG-HA/P is rather difficult, its detailed characterization has not yet been determined. In the present study, we established a new simple purification method for NAG-HA/P. The 419

420

A.

results

shows

kDa

that

purified

ET AL

NAG-HA/Ps

are

in

two

forms

of

34

and

32

proteins. V.

cholerae

Osaka

non-O1

Airport

broth

in

0.01

a 5

liter

flask

sulfate

type

(about

buffer

monoclonal 5

mg

Sepharose

of

4B

of

to

7.0)

remove

0.2

by

M glycine-HCl

method

covery

of

specific Thus

protease

activity

(unit

we

activity

eluate

by

protease a

than

1.

A

typical

activity

(-•›-)

Elution

with

Fractions

indicated

the

of

M glycine-HC1 the

arrow

(•¬)

protein) method

rates

purified

(10),

from using

(pH

2.7) were

an

was used

as

started as

at

purified

the the

about

50

NAG-HA/P technique

the fold. using

is

required dialyzing

analyzed

by

column. substrate

is also

fraction

indicated

NAG-HA/P.

Re-

and

of

immunoaffinity

assayed

27%

This

After was

azocasein

1) (10).

about

which

(1.6%).

NAG-HA/P

(10)

(Fig.

increased

previously

NAG-HAT

buffer

of

chromatography.

recovery

method

mg

was

with

immediately

previously

method

baseline

eluted

was

activity

described

the

was

and

cm)

a column

M phosphate

reached

material

protease as

to

0.01

nm

M NaCl

purification

described

the

profile by

by

per

rapid

8.0),

elution

0.2

purification

lower

(pH

assayed

exhibited

this

methods

with

buffer

0.5

substrate

column

the

bound

(1 •~ 7

applied

with

280

manufacturer's

column

was

at

An (11)

bromide-activated the

a

washed

(10). against

NAG-HA/P

to

into

eluate

soy

NAG-HA/P.

cyanogen

according

was

the

at

tryptic

dialysis

against

packed

diarrhea of

previously

crude

sample)

the

activity

simple,

procedures

M Tris-HC1

was

containing

as

immunoaffinity

complicated

Fig.

The

described

as

of

1 liter

extensive

used

gram

column of

2.7)

azocasein

established

convenient

the

as

developed

Specifically

(pH

of

hr

Sweden)

density

traveler's in

after

were

one

gel

and

optical

M Tris.

the

MAb-coupled more

2

to

materials.

using

C

Chemicals,

4B

the

20

(2-4-2)

with

cultured

sulfate-fractionated

buffer

with

the

C for

4

coupled

(ammonium

until

at

MAb-coupled

non-specific

neutralized

30

patient

and

samples

7.0)

Fine

a

used

(MAb)

Sepharose

(pH

at

was

the

sample

MAb-coupled

buffer

shaking

(pH

protein)

After

crude

from was

antibody

(Pharmacia

instructions. the

with

isolated Station,

(40-55%)-fractionated

M phosphate

IgG

TH81,

Quarantine

Ammonium

0.05

the

NAKA

far

more against SDS-slab

Protease shown. (•«).

NOTES

A

421

B

Fig. 2. Sl)S-slab PAGE (A) and Western blot analysis using polyclonal antibody (B). 1, NAG-HA/P purified by the method described previously (9); 2, NAG-HA/P purified by immunoaffinity column chromatography; 3, molecular weight markers (97.4, 66.3, 42.4, 30.0, 20.1 and 14.4 kDa, respectively).

Fig. 3. Detection of protease activity after conventional (non-denatured) slab-PAGE on a skim milk agar plate. After electrophoresis, the gel was overlaid on skim milk agar to detect the protease activity (10). 1, 32 kDa NAG-HA/P obtained by the method described previously (10); 2, 34 kDa NAG-HAIP purified in this study (activity of a minor 32 kDa protein contained in this preparation did not appear in this condition).

422

A.

NAKA

ET Al.

Fig. 4. Double gel diffusion test. 1, and 32 kDa NAG-HA/P antisera; 3, purified 32 kDa NAG-HA/P; 4, purified 34 kDa NAG-HA/P.

2, crude

NAG-HA/I);

PAGE (8), which showed two protein bands with a molecular weight of about 34 kDa (usually the major band) and 32 kDa (usually the minor band). The latter band seems to be identical (Fig. 2A) with that of NAG-HA/P purified as reported previously (10). These two major proteins , 34 and 32 kDa proteins, were also examined by conventional (non-denatured)-slab PAGE (9) , showing that both 34 and 32 kDa proteins possessed protease activity as demonstrated on a skim milk agar plate (Fig. 3). The 32 kDa protein migrated faster than the 34 kDa (Fig . 3) in non-denatured-slab PAGE. Immunological cross-reactivity of 34 and 32 kDa proteins was demonstrated by Western blot analysis (Fig. 2B) performed as described (9) and double gel diffusion test (Fig. 4) using 1% Noble agar (8) which showed that both proteins formed a fused precipitin line against anti NAG-HA/P antisera against 32 kDa protein (10). Furthermore, Western blot analysis showed that a polyclonal antibody reacted similarly to both 34 and 32 kDa proteins. Identical Western blotting pattern was also obtained with the monoclonal antibody. These results suggest no detectable immunological difference between the 34 and 32 kDa proteins, at least by using antisera against 32 kDa protein. Thus the NAG-HA/P purified by immunoaffinity column chromatography suggests that NAG-HA/P has two forms (or isoforms). One has a molecular weight of 34 kDa which was not purified by the conventional purification procedure (10), and the other has molecular weight of 32 kDa. We are not sure at this moment the relationship between the two proteins. However, the facts that the 34 kDa protein was obtained only by our (immunoaffinity) purification method described here and that the two proteins are immunologically indistinguishable suggest that they are closely related and that the 34 kDa protein may be a preform of the other or an isoform. Further analysis regarding this issue is currently in progress in this laboratory. Several studies have demonstrated the importance of Vc-HA/P in the pathogenesis of V. cholerae (3, 5, 6, 13). Therefore, the simple purification method described in this communication greatly contributes to the use of Vc-HA/P such as a vaccine.

NOTES

423

This work was supported by a Grant-in-Aid for Scientific Research and for Special Project Research from the Ministry of Education, Science and Culture of Japan. REFERENCES

1)

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16)

Yamamoto, K., Al-omani, M., Honda, T., Takeda, Y., and Miwatani, T. 1984. Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin. Infect. Immun. 45: 192-196. Yoh, M., Honda, T., and Miwatani, T. 1986. Purification and partial characterization of a cholerae

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vibrio

52: 319-322.

(Received for publication, October 7, 1991)

protease by immunoaffinity column chromatography using a monoclonal antibody.

A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immun...
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