Journal

of
Microbiological
Journal of Microbiological Methods 29 (1997) 201–206
Methods

A simplified method for isolating outer membrane proteins from

Pasteurella haemolytica A1
a, a b
Robert E. Brennan *, Richard E. Corstvet , Jere W. McBride
a
Department of Veterinary Science, 111 Dalrymple Building, South Campus Dr., Louisiana State University, Agricultural Center, Baton
Rouge, LA 70803, USA
b
Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
Received 5 February 1997; received in revised form 27 May 1997; accepted 27 May 1997

Abstract

The outer membrane of Pasteurella haemolytica A1 (Ph-1) contains several major proteins which may play an important
role in stimulating protective immunity. This report describes a method which was used to successfully isolate three Ph-1
outer membrane proteins (OMPs). The molecular weights of these OMPs are 31 kilodaltons (kD), 40 kD and 42 kD
respectively. This method involves two steps: first, a sarcosyl extraction step was used to obtain a crude OMP prep; and
secondly, these OMPs were separated by gel electrophoresis using a Bio-Rad 491 Prep Cell which separates proteins based
on their molecular weights. Isolation of the proteins was demonstrated by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE). Immunogenicity was then demonstrated by immunoblotting and enzyme-linked immuno-
sorbent assays (ELISAs) with convalescent serum taken from calves with prior exposure to Ph-1. SDS-PAGE results show
that single bands of each protein were obtained. Immunoblots and ELISAs of these proteins show that the convalescent
antiserum used reacted with the 31 kD and 40 kD proteins, but did not react with the 42 kD protein. This procedure provides
for a simplified method to obtain highly purified OMPs from Ph-1 which may now be evaluated on an individual basis as to
what role they may play in stimulating resistance to pasteurellosis.  1997 Elsevier Science B.V.

Keywords: Pasteurella haemolytica A1; Outer membrane proteins; Isolation; ELISA

1. Introduction reduce the prevalence of this disease using live

Pneumonic pasteurellosis has been recognized as a
source of major economic loss to the cattle industry
since the early 1900’s [1–3]. It is now thought that
the primary agent responsible for producing the
fibrinous pneumonia associated with this disease is
Pasteurella haemolytica A1 (Ph-1) [3]. Attempts to
*Corresponding author. Tel.: 11 504 3884769; fax: 11 504
3884890.
vaccines, recombinant vaccines, bacterins, superna-
tant preparations and subunit preparations have had
limited beneficial effects [2]. The inconsistency of
these vaccines can in part be attributed to the
variability of their antigenic content and lack of
knowledge about which antigens are important for
stimulating protective immunity [4,5]. Efforts over
the last ten to fifteen years have been focused on
identifying and characterizing Ph-1 components
which may be important for stimulating protective
immune responses [4]. Among these components are

0167-7012 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved.

PII S0167-7012( 97 )00050-X
202 R.E. Brennan et al. / Journal of Microbiological Methods 29 (1997) 201 – 206
several outer membrane proteins (OMPs). In many
bacteria OMPs are known to play an important role
in the infectious process through their ability to act
as porins and through their involvement in adhesion
of bacteria to host cells [6]. It is thought that
antibodies to these OMPs could serve as effective
immunogens by preventing adherence, promoting
phagocytosis, and causing death of the bacteria by
interfering with membrane function [6]. Several
studies have indicated that antibodies to Ph-1 OMPs
do play a vital role in resistance to pasteurellosis
[5,7,8]. In order to identify which proteins are
responsible for resistance it is necessary to separate
and characterize them [9]. Isolation of these proteins
would also be useful in vaccine production as well as
for evaluating systemic as well as local antibody
responses [10].
Extraction of Ph-1 OMPs was previously reported
by Squire et al. [6]. The OMP fraction produced
contained two major proteins; one at 30 kD and one
at 42 kD. Another study [11] reported that a saline
extracted antigen prep contained a 29 kD and a 44
kD protein that were recognized by Ph-1 antiserum
by immunoblot. These studies demonstrated that
OMPs of Ph-1 are extractable and are immunogenic.
The goal of this project was to use a simple method
to obtain highly purified proteins from Ph-1 OMP
extracts and achieve antigenic yields large enough so
that these proteins can be studied on an individual
basis with respect to their immunogenicity.
2. Materials and methods
2.1. Organism and growth conditions
A lyophilized preparation of Pasteurella
haemolytica A1 (Ph-1), originally isolated from an
infected calf [12], was suspended in phosphate
buffered saline (PBS) pH 7.2 and cultivated on brain
heart infusion agar with 5% bovine blood for 18–24
h at 378C. One isolated colony was picked and
inoculated into 1 l of BHI broth and cultivated for an
additional 18 h at 378C with orbital shaking. Bacteria
were then harvested by centrifugation in 50 ml
centrifuge tubes at 6000 g for 20 min.
2.2. OMP extraction
OMP extraction was performed according to a
method previously described [13]. Briefly,
sedimented bacteria were pooled and suspended in
35 ml of 20 mM Tris (pH 7.2) and pelleted at 6000 g
for 20 min. Bacteria were then resuspended in 35 ml
of 20 mM Tris (pH 7.2) and placed in a 50 ml
beaker and sonicated with 0.750 probe (55 mm,
amplitude setting 9.20 Hz) on ice for 6 min continu-
ously. Sonicated prep was centrifuged at 6000 g for
20 min. The supernate, which contained the cell wall
and OMPs was removed with a pipette and placed
into a clear ultracentrifuge tube and pelleted at
60 000 g for 1 h. The supernate was then removed
and the pellet was resuspended in 1 ml of 20 mM
Tris (pH 7.2). Cytoplasmic membranes were solubil-
ized by adding 4 ml of 0.5% N-lauryl-sarcosine and
incubating at room temperature for 30 min. Clumps
were broken up by pipetting up and down several
times. The OMPs were then transferred to a 5 ml
ultracentrifuge tube and pelleted at 60 000 g for 1 h
and washed once in 20 mM Tris (pH 7.2). The pellet
was then resuspended in 1 ml of 20 mM Tris (pH
7.2) and the protein concentration was determined
using the Bio-Rad DC Protein Assay (Bio-Rad Life
Science, Hercules, CA, USA).
2.3. Isolation of OMPs
Isolation of the individual proteins from this crude
prep was then accomplished using the 491 Prep Cell.
The 491 Prep Cell has previously been used by
Hager et al. and Yamamoto and Munn to successful-
ly isolate proteins from other agents. [14,15]. First,
analytical sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) was used to de-
termine the acrylamide concentration (%T) that
would best separate the 31 kD and the 42 kD
proteins from the nearest contaminants. It was de-
termined that an 11% resolving gel would give us the
best separation. An 11% polyacrylamide resolving
gel was cast into a 27 mm gel tube to 10 ml and
overlaid with 1 ml of water saturated 2-butanol. This
was allowed to polymerize for 3.5 h using a distilled
water (dH 2 O) circulation pump as a coolant. The
butanol was then decanted and the gel was rinsed
 R.E. Brennan et al. / Journal of Microbiological Methods 29 (1997) 201 – 206
two times with dH 2 O. A 4% polyacrylamide stack-
ing gel was cast onto the gel to 14 ml and allowed to
polymerize for 3 h cooled by the circulation pump.
The 1 ml of crude extract containing approximately
13 mg of protein per ml was diluted to 7 ml in SDS
reducing buffer and loaded directly onto the gel
using a 20 ml syringe and a 20 14 gauge needle. The
sample was electrophoresed for 5 h at 10 W constant
power. Seventy 5 ml fractions were collected over a
6 h time period using a 211 Multitrac fraction
collector (LKB Bromma, Sweden).
2.4. Identification and immunoblotting of proteins
Identification of the individual proteins was
achieved by SDS-PAGE. Briefly, 12% poly-
acrylamide gels 0.75 mm thick were cast using a
Mini-Protean II electrophoresis system (Bio-Rad
Life Science). A 10 ml volume of individual fractions
were loaded per lane and electrophoresed for 45 min
at 200 volts. The gels were then stained with
Coomassie brilliant blue R-250 (Ameresco, Solon,
OH, USA) to visualize proteins. Samples containing
the proteins of interest were concentrated into 3 ml
samples using Centriprep 3 concentrators (Amicon,
Beverly, MA, USA).
Immunoblotting was done by performing SDS-
PAGE on isolated proteins as before and then
electrophoretically transferring the proteins to a
nitrocellulose membrane (Optitran, Schleicher and
Schuell, Keene, NH, USA) using a Bio-Rad Tans-
Blot SD Semi-Dry Transfer Cell. The transfer was
run at 18 volts for 30 min. Proteins were reacted
with high titer convalescent bovine anti Ph-1 serum
and probed with peroxidase labeled goat anti-bovine
immunoglobulin G (IgG) (heavy and light) (Kir-
kegaard and Perry, Gaithersburg, MD, USA) diluted
1:750 in PBS. The substrate 4-chloro-1-napthol
(4CN) (Kirkegaard and Perry) was used to reveal
protein–antibody reactions.
2.4.1. Enzyme-linked immunosorbent assay
( ELISA)
Isolated proteins were diluted to 4 mg / ml in 0.1 M
carbonate buffer (pH 9.2) and added at 100 ml / well
to 96 well microtitration plates (Dynatech Laborator-
ies, Chantilly, VA, USA). Plates were sealed in a
203
ziplock bag and placed at 48C for 18–24 h for
fixation. Plates were then washed three times with
dH 2 O1NaCl1tween 20 and blocked with NET (0.1
M NaCl11 mM EDTA110 mM Tris base pH 8.0)1
10% goat serum for 30 min at 378C. After blocking,
plates were washed as before and 100 ml of (1)
convalescent bovine anti-Ph-1 sera or (2) pre-Ph-1
exposure sera was added to appropriate wells and
incubated for 1 h at 378C and then the wells were
washed again. The wells were then probed with 100
ml of peroxidase labeled goat anti-bovine IgG (heavy
and light) (Kirkegaard and Perry) diluted 1:750 in
NET110% goat serum and incubated for 1 h at
378C. After a final washing, 100 ml of ABTS [2-
29azino-di(3 ethylbenzothiazoline sulfone-6)] sub-
strate (Kirkegaard and Perry) was added per well and
allowed to develop for 10 min. Optical densities
(ODs) were determined using an ELISA reader (MR
5000 microplate reader, Dynatech) at a wavelength
of 405 nm.
3. Results
3.1. Purification of proteins
After the sarcosyl extraction step previously de-
scribed we obtained a 1 ml sample which contained
approximately 13 mg of crude OMP. Fig. 1 illustrates
Fig. 1. Analysis by SDS-PAGE and Coomassie brilliant blue
R-250 staining of the outer membrane prep after sarcosyl ex-
traction step. Lane 1, Ph-1 whole cell organism (20 mg of protein);
Lane 2, Ph-1 outer membrane proteins (20 mg of protein).
204 R.E. Brennan et al. / Journal of Microbiological Methods 29 (1997) 201 – 206

the contrast between whole cell Ph-1 and what

proteins remain after sarcosyl extraction of the outer
membrane. The two major proteins of interest are
present in this sample as well as other minor
proteins. We were able to separate the 31 kD and 42
kD proteins from the others by preparative gel
electrophoresis using an 11% SDS-PAGE in a 491
Prep Cell (Bio-Rad). We were able to obtain two
distinct and highly pure samples as seen in Fig. 2.
The 31 kD protein was present in fraction numbers
14, 15, and 16, while the 42 kD protein was present
in fraction numbers 34, 35, and 36. We obtained a
final volume of 3 ml for each sample with protein
concentrations of 300 mg / ml for the 31 kD protein
and 500 mg / ml for the 42 kD protein. In the process
of purifying these two major proteins a 40 kD minor
protein was also purified as shown in Fig. 2. This
protein was present in fraction numbers 30, 31, and
32. A concentration of 420 mg / ml of this protein
was recovered.
Fig. 3. Immunoblot analysis of isolated proteins after SDS-PAGE
and transfer to nitrocellulose membrane. Samples were probed
with polyvalent antibody to Ph-1. Lane 1, Ph-1 whole cell; Lane 2,
3.2. Immunoblotting of proteins 31 kD protein; Lane 3, 40 kD protein; Lane 4, 42 kD protein.

The anti-Ph-1 convalescent serum used to probe

these purified proteins reacted with the 31 kD and 40 kD proteins but not the 42 kD protein as shown in
Fig. 3.

3.3. ELISA

Antibody responses obtained by ELISA from the

convalescent (1) and the pre-exposure (2) serum to
the 31 kD and 40 kD proteins are expressed in ODs.
The ODs of the (1) serum to these two proteins
were significantly greater ( p,0.05) than the (2)
serum. The (1) serum had ODs of 1.497 and 1.538
respectively while the (2) serum ODs were 0.476
and 0.623. Results are illustrated in Fig. 4.

4. Discussion

This report has described a two-step procedure for

Fig. 2. Analysis by SDS-PAGE and Coomassie brilliant blue
R-250 staining of the outer membrane proteins after isolation with
Bio-Rads 491 Prep Cell. Lane 1, molecular weight marker (Bio-
Rad); Lane 2, Ph-1 whole cell (5 g of protein); Lane 3, 31 kD
protein (3 mg of protein); Lane 4, 40 kD protein (4 mg of protein);
Lane 5, 42 kD protein (5 mg of protein).
the purification of Ph-1 OMPs which provides us
with the ability to obtain highly purified and immu-
nologically stable proteins. Advantages that we feel
this technique has over other techniques that have
been used to extract proteins from Ph-1 such as
 R.E. Brennan et al. / Journal of Microbiological Methods 29 (1997) 201 – 206
Fig. 4. Comparison between (2) serum ODs and (1) serum ODs to the 31 kD and 40 kD isolated proteins. Results obtained by ELISA.
sucrose density gradients, isoelectrofocusing, and
chromatofocusing [6,11] are the ability to obtain
purer proteins, achieve higher antigenic yields, and
be less laborious. The proteins purified in this
manner were recovered in quantities that will allow
for use in biological assays (i.e. ELISAs and Western
blots). Also, there are several other studies which
have proposed that OMPs from a number of organ-
isms may be suitable components for effective
vaccines [16–18], which may make this technique
even more desirable. One drawback that we ex-
perienced during this study was the amount of time it
took to identify which fractions contained the pro-
teins of interest which can be attributed to our
inability to monitor the proteins as they were eluted
from the 491 Prep Cell. This resulted in the screen-
ing of all of the fractions until we identified which
ones contained the relevant proteins. The incorpora-
tion of a UV monitor and a chart recorder would
have allowed us to determine which fractions con-
tained protein and which ones did not, ultimately
reducing the number of fractions needing to be
screened.
As mentioned before it will now be possible to
begin to investigate, on an individual basis, the
involvement of these OMPs as well as other Ph-1
proteins in the pathogenesis of and the resistance to
Ph-1. Currently two of the OMPs that we have
isolated (31 kD and 40 kD) are being incorporated
into ELISA and Western blot assays in our lab. This
does not suggest that the 42 kD protein is not
important; only that it requires further investigation.
205
The lack of antibody response to this protein as
measured by immunoblot and ELISA may be due to
the fact that the immune system in the animal from
which we collected the convalescent sera did not
recognize this protein. It may also be that this protein
is not an immunologically dominant antigen or that it
may be part of a highly conformational protein
which when denatured during SDS-PAGE is no
longer immunologically active.
In conclusion, this study has described a simplified
method with which we were able to isolate OMPs
from Pasteurella haemolytica A1. The 31 kD and 40
kD proteins are being used to screen samples (nasal,
lavage, serum, and tonsil) from calves that have been
experimentally exposed to Ph-1 and to look at the
local antibody responses to these proteins and how
they may correlate to resistance from pasteurellosis.
These studies could provide valuable information in
the effort to develop new and improved vaccines in
which the identification of which components of
Ph-1 stimulate protective immunity appears to be
crucial.
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