A Rapid Selective Extraction Procedure for the Outer ...

Protein Expression and Purification 25, 134–137 (2002)
doi:10.1006/prep.2002.1619

A Rapid Selective Extraction Procedure for the Outer
Membrane Protein (OmpF) from Escherichia coli

Steven Arcidiacono,* Michelle M. Butler,† and Charlene M. Mello*,1

*U.S. Army Soldier Biological Chemical Command, Natick Soldier Center, Kansas Street,
Natick, Massachusetts 01760; and †Microbiotix, Inc., One Innovation Drive, Worcester, Massachusetts 01605

Received July 12, 2001, and in revised form December 26, 2001

Porins are essential pore-forming proteins found in fusion of low-molecular-weight solutes across the mem-
the outer membrane of several gram-negative bacteria. brane. A number of studies have been conducted to
Investigating the relationships between molecular obtain detailed structural information (1, 2), elucidate
structure and function involves an extremely time-con- the role of lipopolysaccharides (LPS)2 in protein folding
suming and labor-intensive purification procedure. We and channel formation (3, 4), and define solute trans-
report a method for rapid extraction of the outer mem- port (5, 6). Unfortunately, purified porin proteins are
brane protein, OmpF, from freeze-dried Escherichia required to perform these structural and functional
coli cells using valeric acid, alleviating the effort and studies. Despite multiple reports in the literature, the
time in sample preparation. Extraction results in a purification of porin proteins is often lengthy and labor
highly enriched fraction containing OmpF as 76% of intensive, requiring long incubations and multiple ex-
the total protein content. The apparent molecular mass traction steps to simply obtain a porin-enriched fraction
determined by SDS–PAGE mobility was 38,900, similar (2, 7–12). For example, solubilization of E. coli OmpF
to that of the monomeric form of OmpF. N-terminal is achieved by differential extractions including several
sequencing yielded 23 amino acids with 100% identity preextraction steps with detergent to solubilize unre-
to the published OmpF sequence. The trimeric form lated proteins, followed by five consecutive extractions
of OmpF was observed in unheated samples run on with 3% octyl(polydisperse)oligooxyethylene for an en-
SDS–PAGE and analysis of these samples by periodic riched porin sample (8). Final purification of Omp is
acid/silver staining revealed the presence of unbound achieved by gel-filtration and ion-exchange chromatog-
lipopolysaccharides. Furthermore, this method should raphy or continuous flow electrophoresis (2, 10, 11).
prove useful for isolating other outer membrane pro-
teins. ᭧ 2002 Elsevier Science (USA) We report a single-step method for the enrichment
of OmpF from E. coli cells that can be completed in 1
Key Words: outer membrane protein; porin; OmpF; to 2 h. Selective solubilization of OmpF in valeric acid
membrane channels; organic acid extraction. is possible due to its unusually stable trimeric complex.
Many investigators have reported that porin trimers
Porins play a major role in the physiology of gram- are resistant to proteases, detergents, organic solvents,
negative bacteria (e.g., Escherichia coli, Klebsiella a wide variation of pH ranging from 2 to 13, and temper-
pneumoniae, and Pseudomonas aeruginosa). In general, atures up to 70ЊC (8). The strong hydrogen-bonding,
porins exist as tightly bound trimeric complexes that electrostatic, and hydrophobic interactions that stabi-
create water-filled channels facilitating the dif- lize the OmpF trimer are not disrupted in valeric acid.
OmpF-enriched fractions prepared with this method
contain approximately 76% OmpF with associated LPS.

1 To whom correspondence should be addressed at the U.S. Army 2 Abbreviations used: Omp, outer membrane protein; LPS, lipopoly-
saccharides; SDS, sodium dodecyl sulfate; PAS, periodic acid/silver
nitrate stain; BLAST, Basic Local Alignment Search Tool; PIR, Pro-
tein Identification Resource.

134 1046-5928/02 $35.00
᭧ 2002 Elsevier Science (USA)
All rights reserved.

OUTER MEMBRANE PROTEIN EXTRACTION FROM Escherichia coli 135

MATERIALS AND METHODS FIG. 1. (A) Sypro red-stained 10–20% Tricine SDS–PAG of OmpF
from valeric acid lysate. Lane 1, Mark 12 molecular weight standards
Preparation of an OmpF-enriched extract. E. coli (Novex); lane 2, valeric acid E. coli crude fraction, boiled 5 min, 95ЊC
SG13009 pREP4 (QIAgen, Inc., Chatsworth, CA) were in the presence of SDS. (B) Densitometry of valeric acid extract of
grown to midlog in 4ϫYT medium (LϪ1: 32 g tryptone, E. coli cells on Sypro red-stained 10–20% Tricine SDS–PAG. The large
20 g yeast extract, 5 g NaCl) containing 25 ␮g/ml kana- peak corresponds to the OmpF monomer migrating at an apparent
mycin. Cells were harvested by centrifugation, frozen molecular mass of 38,900. This peak represents 76% of the total area
at Ϫ80ЊC, and lyophilized. Lyophilized cells were of all peaks.
ground to a powder with a mortar and pestle and lysed
with 2 ml of valeric acid per gram of lyophilized powder. protein/g dry cell pellet or 2.1 mg of OmpF/g dry cell
Milli-Q water was used to dilute the mixture fourfold weight calculated from the densitometric purity deter-
to a final valeric acid concentration of 2.3 N. The sample mination. To date, this is the most rapid OmpF enrich-
was stirred for 1 h at room temperature and centrifuged ment procedure and yields the highest OmpF purity (2,
at 20,000g for 10 min at room temperature to remove 11). This method may prove to be a useful tool in the
cell debris. purification of porin proteins for detailed structural and
functional studies.
SDS–PAGE. Clarified supernatants were run on
10–20% Tricine SDS–polyacrylamide gels (Invitrogen N-terminal sequencing analysis was performed to
Corp., Carlsbad, CA). The samples were run either un- identify the 38,900-Da protein band and resulted in
heated or heated (5 min at 95ЊC) in Laemelli sample the sequence AEIYNKDGNKVDLYGKAVGLHYF. A
loading buffer (25 mM Tris–Cl, 2% SDS, 0.1% bromphe- BLAST search of the PIR database identified a number
nol blue, 10% glycerol). Proteins were stained with Coo- of sequence alignments with porin proteins at an 80%
massie Blue R-250 or SYPRO red (Molecular Probes, or greater similarity. The E. coli OmpF protein (PIR
Eugene, OR) for 30 min in 7.5% acetic acid and de- accession MMECF) sequence (14) yielded 100% similar-
stained for 10 min with 7.5% acetic acid. SYPRO red- ity at the amino acid level to the N-terminal sequence
stained gels were visualized on a Storm 860 optical data. An alignment of this published sequence and the
scanner (Molecular Devices, Sunnyvale, CA) using the N-terminal sequence data are depicted in Fig. 2.
red fluorescence/chemifluorescence mode at 800 V. Pro-
tein purity was determined by densitometry with Image The quantity and purity of OmpF in the crude extract
QuaNT software (Molecular Devices). The baseline was was dependent on the volume of valeric acid used. Not
established by drawing a straight line between the two all of the OmpF was extracted with the initial cell lysis.
lowest valleys on the graph. LPS was detected by peri-
odic acid/silver nitrate (PAS) staining (13). Briefly, gels
were fixed and treated with 0.7% periodic acid, followed
by silver staining with a Sigma AG25 silver stain kit.

Characterization of OmpF-enriched fractions. The
monomer band in an SDS–polyacrylamide gel was elec-
troblotted onto a PVDF membrane and N-terminal se-
quencing was performed on a PE/Applied Biosystems
Procise 492. A Basic Local Alignment Search Tool
(BLAST 2.0) search of the Protein Identification Re-
source database (PIR database) (National Center for
Biotechnology Information, NLM/NIH) was used to
identify the protein. Protein quantitation was deter-
mined with the Bio-Rad Protein Assay (Hercules, CA)
using bovine serum albumin as a standard.

RESULTS AND DISCUSSION

Direct lysis of E. coli cells with valeric acid results FIG. 2. Alignment of E. coli outer membrane porin precursor with
in a highly enriched porin sample. OmpF monomer is N-terminal sequence results. There is a 100% similarity to the pub-
the major protein band visualized in an SDS– lished E. coli OmpF protein (PIR accession MMECF) sequence. Resi-
polyacrylamide gel, migrating at an apparent molecular due 1 of the N-terminal sequence aligns with residue 23 of the pub-
mass of 38,900 (Fig. 1A). This band represented 76% lished OmpF precursor sequence. Residues 1–22 from the published
of the protein as determined by densitometry (Fig. 1B). sequence of the precursor represent a signal sequence (14) not found
Total protein content of the OmpF-enriched fraction in the functional OmpF protein.
was determined by Bio-Rad protein assay to be 2.7 mg

136 ARCIDIACONO, BUTLER, AND MELLO

Additional OmpF and other contaminating proteins hydrogen bonding and hydrophobic interactions within
were recovered with repeated valeric acid washes. the trimer, a heterogeneous population of LPS with an
Therefore, the purity of OmpF in the enriched fraction estimated molecular mass between 100,000 and
was achieved with some loss of the total available OmpF 200,000 is released. Areas corresponding to the mono-
protein. There was no evidence of the trimer form in mer and trimer bands are “negatively” stained. The
the initial lysate or any of the washes based on SDS– moieties within LPS that react with this silver staining
PAGE (see Fig. 1A); however, the sample was heated method are thought to be the core polysaccharides (13).
in the presence of SDS prior to electrophoresis. The If LPS were bound to the monomer or trimer bands, it
presence of monomer after heating is consistent with did not react with the carbohydrate-specific silver
previously published results for outer membrane pro- stain. This may be due to an insufficient concentration
teins (2, 11). Porin trimers are stable in SDS, but will of LPS or the masking of these reactive groups, which
dissociate and migrate as monomers when heated to would result in an inhibition of silver staining. It is
100ЊC. When the sample was loaded without heating, therefore difficult to identify the presence of any tightly
a band corresponding to the trimeric complex appeared bound LPS (see Fig. 3B). Many OmpF-enriched frac-
with an apparent molecular mass of 63,100 (Fig. 3A). tions have LPS in bound and free forms. The free form
This is well below the relative mass at which the trimer is often removed by gel-filtration chromatography and
would be expected to migrate. However, identification the bound form is largely removed with ion-exchange
of outer-membrane proteins based on their electropho- chromatography and ultrafiltration. Both forms of LPS
retic mobility in SDS–PAGE has often been misleading are generally visible by silver staining (13). However,
due to anomalous mobility (11). Perhaps the presence of it is still a matter of discussion how critically the struc-
acid prevents full trimer formation or results in altered ture and function of porins depend on the presence of
electrophoretic migration. LPS (7).

Analysis of OmpF-enriched fractions by SDS–PAGE Traditional purification of porin proteins entails iso-
with Coomassie blue and periodic acid/silver staining lation of a crude porin/membrane extract and subse-
reveal the presence of unbound LPS (Fig. 3). Bands quent steps to remove LPS and contaminating proteins.
visible by PAS stain, migrating at relative molecular We have demonstrated a one-step method for the en-
masses between 6000 and 36,000 do not correspond to richment of OmpF from E. coli by extraction with va-
any protein bands seen on the Coomassie blue-stained leric acid that takes place at room temperature with
gel (Fig. 3A). They appear to be free forms of LPS not basic laboratory equipment and can be completed in 1
directly associated with the OmpF protein. Upon heat- to 2 h. Further purification steps were not attempted.
ing the sample and presumably disrupting the strong However, chromatographic (gel filtration and ion ex-
change) and electrophoretic separations developed for
several outer membrane proteins (2, 7, 8, 12, 13) may
be utilized with the reported valeric acid extraction
procedure to yield a highly purified protein sample.
Finally, OmpF has significant similarities with other
outer membrane proteins, which suggests that this
method may be useful for isolating different porins from
other gram-negative bacteria.

ACKNOWLEDGMENTS

We thank the BioResource Center (Cornell University) for amino
acid analysis and N-terminal sequencing. We are also grateful to Dr.
Alfred Allen, Robert Stote, and Anthony Lombardo (Natick Soldier
Center) for valuable input and discussions.

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