Protein Expression and Purification 131 (2017) 16e26

Contents lists available at ScienceDirect

Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Purified Stx and l phage initiator O proteins bind specifically to two

different origins of replication in vitro
Katarzyna I. Kozłowska*, Joanna Tymecka-Mulik, Grzegorz We˛grzyn

sk, Poland
Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308, Gdan

a r t i c l e i n f o a b s t r a c t

Article history: The O protein is a crucial factor initiating the DNA replication of lambdoid bacteriophage. Efficient DNA
Received 30 August 2016 replication of Shiga toxin-converting phage is necessary for effective production of Shiga toxin e main
Received in revised form virulence factor of STEC strains. We developed an improved protocol for overproduction, bacterial cell
24 October 2016
lysis and purification of lO protein. With use of this method we have also isolated O proteins of Stx-
Accepted 4 November 2016
phage P27 and 933W that were never purified before. Purified proteins were tested for their DNA
Available online 5 November 2016
binding activity and revealed a sequence specific interactions.
© 2016 Elsevier Inc. All rights reserved.
Keywords:
Protein overproduction
Protein purification
Initiator O protein
DNA-Protein interactions
l phage
Stx-phage

1. Introduction studies on the mechanisms of processes such as phage induction or

Escherichia coli naturally inhabits the human digestive system
and is an important part of the digestion process [1]. Most E. coli
strains are commensals, but some may be very dangerous patho-
gens. They include Shiga toxin producing E. coli (STEC) and a subset
of these strains called enterohemorrhagic E. coli (EHEC). Infections
caused by these micro-organisms may lead to severe complications
like: bloody diarrhea, haemolytic uraemic syndrome (HUS) or
hemorrhagic colitis (HC), often leading to death. They are especially
dangerous for children and elderly people [2e4]. The main viru-
lence factor of STEC strains is Shiga toxin, encoded by Shiga tox-
ineconverting prophage, or Stx-phage, belonging to the lambdoid
family [5]. Efficient production of Shiga toxin requires the induction
of prophage followed by its DNA replication and lytic development
in the host cell [6]. STEC were responsible for a number of out-
breaks including the recent one in Europe (mostly in Germany),
which resulted in over 4000 severe cases [7,8]. Treatment of the
STEC infections is very problematic, due to stimulation of prophage
induction by many antibiotics and antimicrobial agents, which
result in an intensified production of the toxin [4,9]. Therefore,
* Corresponding author.
E-mail address: katarzyna.kozlowska@biol.ug.edu.pl (K.I. Kozłowska).
DNA replication of the Stx-phage are very important, as they may
lead to better understanding of the basic control processes of the
lambdoid phage development and pathogenicity of STEC strains as
well as facilitate the design of new methods of detection, preven-
tion and therapy of these infections.
Stx-bacteriophage belong to lambdoid family which possesses a
similar genome structure resembling that of l bacteriophage [6].
Therefore, progress of biological processes of Stx-phage is probably
congruent with l phage, though never thoroughly studied before.
DNA replication of l bacteriophage depends on complex in-
teractions of the phage-encoded specific replication proteins with
the host replication apparatus [10]. To initiate l phage DNA repli-
cation in vitro, the system of nine proteins is required. Only two of
these proteins are encoded by the phage, while others are provided
by the bacterial host [11]. Viral proteins taking part in the DNA
replication initiation are products of o and p genes, which tran-
scription is controlled by the pR promoter.
l phage DNA replication starts at oril located within the o gene.
The origin consists of four imperfect palindromes of 18e19 bp,
called iterons, and a region rich in AT pairs of 40 bp length [12]. DNA
replication process is initiated by the O protein, which recognizes
and bind in a dimeric form, to the negatively supercoiled DNA of the
specific iteron sequences [13]. The O protein dimers bind firstly to
the two middle iterons and then to two external. Bound proteins

http://dx.doi.org/10.1016/j.pep.2016.11.002
1046-5928/© 2016 Elsevier Inc. All rights reserved.
 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26
oligomerize into a structure called “O-some”, in which DNA is most
likely wrapped around the protein core in a similar way as in
eukaryotic nucleosome. Binding of four protein dimers causes DNA
bending, and produces in this manner thread tension facilitating
the melting of the AT-rich region [12]. The formation of the “O-
some” complex alters the conformation of the origin and this
structure serves as a landing pad for phage P protein which delivers
the host helicase e DnaB [14]. Preprimosomal complex, containing
oril, O, P and DnaB is formed, and later stages of DNA replication
initiation can occur. Formation of the replication complex primes
several rounds of the early l DNA replication that proceeds in
accordance to the circle-to-circle (q) mode [15].
Although sequences of the O initiator proteins among members
of the lambdoid family are conserved, there are some differences in
their amino acid composition that may have a considerable impact
on the functionality of the replication apparatus in various phage.
Previously discovered changes between O proteins of l and Stx-
phage, including P27 and 933W phage, are one or two amino acid
substitutions and two additional iterons [9]. It was found that the O
proteins derived from bacteriophage P27 and 933W have amino
acid substitution (Leu37Ile) in the N-terminal domain responsible
for interactions with DNA. The protein encoded by 933W phage has
also a substitution (Ser282Gly) in the C-terminal domain which
interacts with the P protein [9]. There is another alteration between
lO and Stx-phage O proteins, namely, at position 17 glycine (lO) is
substituted by glutamic acid in phage P27 and 933W. These dif-
ferences may underline changed regulation of the DNA replication
process of Stx-phage compared to l phage. Altered control of this
process was shown to be associated with the degree of influence of
transcription on the DNA replication initiation of these phages [16].
In vivo l phage DNA replication initiation is dependent on
transcription. It was shown that transcription starting from the pR
promoter, ~1000 bp upstream of oril, is necessary to initiate
multiplication of l DNA [17]. This mechanism was called “tran-
scriptional activation” of the origin. Although molecular mecha-
nism of this process still remains unclear, it is probably associated
with DNA supercoiling introduced by RNA polymerase in an active
transcription process - positive in front of the enzyme, and negative
behind it [18]. These changes in DNA topology may not only facil-
itate DNA melting by lowering the energy needed for strand sep-
aration, but also can play a key role in modeling DNA-protein and
protein-protein interactions in the replication complex. Transcrip-
tion starting from the pR promoter might enable lO protein to bind
efficiently to negatively supercoiled DNA and to bend it, which
results in a formation of structurally proper “O-some” complex.
Moreover, recent studies provided evidence for a direct interaction
between RNA polymerase b subunit and the lO protein, which
in vitro enhanced formation of “O-some” [18]. The O proteins
derived from Stx-phage appear to be independent on such assis-
tance with forming an operative “O-some” complex. Plasmid
replicons derived from Stx-phage including P27 and 933W, con-
trary to l plasmid, could replicate in E. coli dna46 strain which has
decreased transcriptional activity [16]. DnaA protein is a stimu-
lating factor for transcription from the pR promoter. Its mutated
equivalent e Dna46 is temperature sensitive and has a very low or
even no DNA binding activity at 43
C. Results of this study indicate
that replication of these lambdoid plasmids is DnaA independent,
moreover it may be transcription independent [16].
Different control of the DNA replication initiation may influence
efficiency of production of Shiga toxin under different conditions in
bacterial cell. Thus, it is crucial to find factors responsible for this
alteration and to resolve its mechanism.
The first step towards understanding the changed regulation of
the DNA replication process is to resolve whether it is caused by
differences in the initiator proteins' structure. To accomplish this, it
17
is necessary to purify different variants of Stx-phage O proteins in a
stable active form suitable for molecular in vitro analyses.
Efficient overproduction and purification of the lO protein has
been very problematic so far. It was demonstrated that in vivo, lO
protein is very unstable with approximately 1.5 min of half-life [19].
The free-form O protein instability in E. coli cells is associated with
its rapid degradation by the host-encoded ClpP/ClpX protease
[20,21]. This proteolysis makes the O protein the main limiting
factor for the l DNA replication initiation in bacteria with low
growth rate [22] and strongly hinders effective overproduction of
this initiator. lO protein also has a tendency to aggregate [23]
which is another obstacle in attempts of its purifications. Due to
changed amino acid composition, Stx-phage O proteins isolation
may be even more problematic.
We managed to overcome these obstacles and developed a
simple protocol for efficient overproduction and purification of Stx-
phage O proteins as well as lO protein. The bacterial lysis procedure
used was a modification of the method described by Roberts and
McMacken [24]. His-tagged proteins were isolated to near homo-
geneity by a two column technique using AKTA € Protein Purification
System. We have also investigated purified O proteins' activities
towards their binding to the l and Stx-phage origins of the DNA
replication by a simple EMSA test.
2. Materials and methods
2.1. Software
For nucleotide and amino acid alignments construction, we used
Bioedit software e a free biological sequence alignment editor that
is available at http://www.mbio.ncsu.edu/. Online Basic Local
Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/) was
used for checking correctness of sequences that we own, infer
functional and evolutionary relationships between sequences as
well as to place them in their gene family. Densitometric analyses of
protein gels' images were carried out using Quantity One 4.6.6.
program (http://www.bio-rad.com/). For isoelectric point calcula-
tions, we used online tool - Isoelectric Point Calculator available at
http://isoelectric.ovh.org domain.
2.2. Bacterial strains and plasmids
Escherichia coli DH5a and Rosetta (DE3) pLysS, with chloram-
phenicol resistance gene, as well as plasmids: pET24a - kanamycin
resistant [25], pUC18 - ampicillin resistant [26], pCB104cmr - l
phage derived, chloramphenicol resistant, P27cmr - P27 phage
derived, chloramphenicol resistant, and 933Wcmr - 933W phage
derived, chloramphenicol resistant [9] were obtained from strain
and plasmid collection of the Department of Molecular Biology of
University of Gdansk.
2.3. Antibodies
For cross-linking assay we used polyclonal rabbit antibodies
against the lO protein [27]. Secondary HRP-conjugated anti-rabbit
antibodies were obtained from Sigma-Aldrich.
2.4. Plasmid construction
Plasmids for O proteins' overproduction were constructed using
standard methods of molecular cloning [28]. O genes from plasmids
pCB104cmr, P27cmr and 933Wcmr [16] were amplified with the use
of primers: lamO_NdeI - AGGAGTCCATATGACAAATACAGCAAAAA-
TACTCAACTTCGGCAGAGG and lamO_his - GCGAAGCTTTCAGTG-
GTGGTGGTGGTGGTGTAGATCCACCCCGTAAATCCAGTCTGT. Primer
18 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26
 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26 19

Fig. 2. Alignment created using Bioedit software, showing changes in amino acid sequences of initiator O proteins of l phage and two Stx-phage: P27 and 933W. 13 additional
amino acids in Stx-phage sequences located at residues from 117 to 129, amino acid substitutions in N-terminal domains (interactions with DNA) of phage P27 and 933W, and in C-
terminal domain (interactions with protein P) of 933W phage O protein are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

lamO_his contained 6x His-tag sequence. PCR products and plasmid
pET24a were cut using restriction enzymes NdeI and HindIII
(Thermo Scientific™). Amplified o genes were ligated with pET24a
plasmid fragments, placing them in the multiple cloning sites under
control of T7 promoter. Following transformation of E. coli DH5a
with selection for kanamycin resistance, plasmid DNA was isolated
from single colonies in accordance with GenElute™ Plasmid Mini-
prep Kit (Sigma-Aldrich) protocol, and sequenced. For further pro-
cedures, we chose a couple of properly constructed plasmids with
correct o genes nucleotide sequences. Obtained plasmids were
named pET24a_wtO_histag, pET24a_P27O_histag and pET24a_
933WO_histag. Plasmids for EMSA test assays, containing l and Stx-
phage origins of DNA replication, were constructed in the same
matter. DNA fragments from plasmids pCB104cmr and P27cmr were
amplified using: oriL_pUC_for_BamH - TCGAGGATCCGACCAAGC-
GACAGTTTAAAG and oriL_pUC_rev_PstI - TGCACTGCAGATTCGGT-
TTTCTGGCTGATG primers. PCR products and plasmid pUC18 were
then cut using restriction enzymes BamHI and PstI (Thermo Sci-
entific™). Following ligation of DNA fragments with plasmids and
transformation of E. coli DH5a with selection for ampicillin resis-
tance, plasmid DNA was isolated from single colonies and verified
by sequencing. Obtained plasmids were named pUC18_oril and
pUC18_oriStx.
2.5. O proteins purification
yeast extract, 1% NaCl). Bacteria were then cultured with shaking at
37
C until they reached OD600 of 1. Following addition of IPTG to final
concentration of 1 mM, bacteria were cultured for another 2e3 h.
Suspensions were cooled on ice and centrifuged for 15 min at
4000 rpm at 4
C. From 1 L of the culture, we obtained 3e6 g of
bacterial cell paste. The paste was washed with P1 buffer (50 mM
Tris-HCl pH 7.6, 10% saccharose), suspended and frozen for overnight
in P1 buffer in ratio 1 paste gram per 2 mL of the buffer.
2.5.2. Bacterial cells lysis
We performed a gentle bacterial cell lysis to avoid degradation
of unstable O proteins. 6 g of bacterial cells paste was slowly
thawed on ice. Then, 15 ml of lysis buffer with spermidine (100 mM
Tris-HCl pH 7.6, 0.5 M NaCl, 10 mM DTT, 100 mM (NH4)2SO4, 10 mM
spermidine trichloride) and 7 ml of 10 mg/ml lysozyme were
added. The solution was complemented with P1 buffer to final
volume of 100 ml. Incubation on ice was conducted for 40 min with
gentle stirring from time to time. Nonionic detergent Triton® X-100
was added to a final concentration of 0.1% in the case of Stx-phage O
proteins purification (without this detergent we could not obtain
sufficient amount of native proteins). Heat shock was performed at
43
C for 5 min. This procedure was repeated two times for lO
protein and three times for Stx-phage O proteins, with 15 min in-
cubation on ice with gentle stirring between each heat shock. The
lysate was centrifuged for 60 min at 19,000 rpm.

2.5.1. Overproduction of O proteins 2.5.3. Ammonium sulfate fractionation

E. coli Rossetta strain was transformed with plasmids: pET24a_ The supernatant was fractionated with 0.22 g per 1 ml ammo-
wtO_histag, pET24a_P27O_histag and pET24a_933WO_histag, and nium sulfate (38% ammonium sulfate saturation). Ammonium
cultured overnight in LB medium at 37
C. Overnight cultures were sulfate was added slowly for about 30 min at 4
C with gentle
rejuvenated in 1:50 ratio into 4 L of LB medium (1% tryptone, 0.5% stirring. Then, suspension was centrifuged for 60 min at

Fig. 1. Alignment created using Bioedit software, showing differences in nucleotide sequences of the o genes of l phage and two Stx-phage: P27 and 933W. Iterons e sequences
recognized by O proteins during the initiation of the DNA replication, are marked in yellow. Substitutions in codons for amino acids located in N-terminal domains (interactions with
DNA) of O proteins of phage P27 and 933W, and in C-terminal domain (interactions with protein P) of p933W phage are highlighted in red. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
20 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26

19,000 rpm. Supernatant was discarded, and the sediment was at 4
C, 80 V for 3.5 h. Gel was incubated for 30 min in 0.1 mg/ml
dissolved in 0.1 volume of the original supernatant of O1 buffer ethidium bromide solution with gentle rocking. Results were
(50 mM Tris-HCl pH 7.9, 20% glycerol, 0.1 M NaCl, 1 mM EDTA, 5 mM visualized on a Gel Doc XR þ scanner (Bio-Rad).
DTT, 0.02% Triton® X-100). Next step was performed as previously
described, but with 0.24 g of ammonium sulfate per 1 ml (40%
2.6.2. Disuccinimidyl glutarate cross-linking assay
ammonium sulfate saturation). Precipitate was collected again by
Protein cross-linking assay was performed with the use of cross-
centrifugation for 45 min at 19,000 rpm. Sediment was dissolved in
linking agent disuccinimidyl glutarate (DSG) (Sigma-Aldrich). 200 ng
5 ml of O2 buffer (20 mM sodium phosphate pH 7.4 [pH 6.5 for l O
of plasmid was incubated with 1 mg of O protein in an O incubation
protein], 0.1 M NaCl, 10% glycerol, 0.1 mM EDTA, 6 mM b-mercap-
buffer, in a final volume of 20 ml, for 15 min at 4
C. 10 ml of DSG
toethanol, 0.02% Triton® X-100) and applied on two desalting col-
solution in DMSO (0.4 mg/ml) was then added, and incubation was
umns (2.5 ml load) Sephadex G-25 M (GE Healthcare, 8.3 ml bed
carried out for additional 30 min. Cross-linking reaction was stopped
volume). Desalting on a column was executed according to the
by addition of Tris-HCl pH 7.5 solution to a final concentration of
procedure manual. We obtained an eluate in a 7 ml of Binding
0.2 M. Samples were incubated for 15 min at room temperature,
buffer (20 mM sodium phosphate pH 7.4 [pH 6.5 for lO protein],
10% glycerol, 3 mM b-mercaptoethanol, 0.5 M NaCl, 20 mM imid-
azole, 0.02% Triton® X-100 [was not added in case of lO protein]).

2.5.4. Purification on a HisTRAP column

€
To purify O proteins, we used AKTA Fast Protein Liquid Chro-
matography (FPLC) system. Eluate from the previous step was
added onto a 1-ml HisTRAP HP column (GE Healthcare), previously
equilibrated with Binding buffer. Column was washed with 10
column volumes of Binding buffer at flow rate 1 ml/min. Next steps
were: washing with 5 vol of 10% Elution buffer (20 mM sodium
phosphate pH 7.4 [pH 6.5 for l O protein], 10% glycerol, 3 mM b-
mercaptoethanol, 0.5 M NaCl, 0.5 M imidazole, 0.02% Triton® X-100
[was not added in case of lO protein]), 5 vol of 20% Elution buffer,
5 vol of 30% Elution buffer. Finally, the column was washed with
15 vol of 30e100% gradient of Elution buffer. Fractions were elec-
trophoresed in denaturing 12% polyacrylamide gels in TGS buffer.
Proteins were visualized by colloidal Coomassie Brilliant Blue gel
staining procedure, as described by Dyballa et al. [29].

2.5.5. Dialysis
Fractions with eluted O protein were combined and dialyzed
against 3 l of Dialysis buffer (0.2 M sodium phosphate pH 7.4 [6.5
for lO protein], 0.5e0.125 M NaCl, 10% glycerol, 1 mM DTT, 0.02%
Triton® X-100 [it was not added in case of lO protein]) in three
steps with decreasing concentrations of sodium chloride, overnight
at 4
C. This step was necessary to avoid protein precipitation due to
rapid reduction of sodium chloride concentration.

2.5.6. Size-exclusion chromatography

After the dialysis, the solution was added into a gel filtration
column Superose 12 10/300 GL (GE Healthcare), and filtration was
performed with Dialysis buffer without addition of Triton® X-100,
at 0.5 ml/min flow rate. In case of the O protein derived from 933W
phage, this step was repeated. Fractions with purified O protein
were combined, complemented with glycerol to a final concen-
tration of 50%, sodium chloride to 0.1 M, and frozen in liquid ni-
trogen. Proteins were stored at 80
C. Total protein concentrations
in samples were determined with the use of FluoroProfile® Protein
Quantification Kit (Sigma-Aldrich), accordingly to the manual.

2.6. Experimental procedures

2.6.1. EMSA assay

Electrophoretic mobility shift assay (EMSA) was executed in a
freshly prepared O incubation buffer (0.2 M sodium phosphate pH
7.4, 0.1 M NaCl, 0.1 mM EDTA, 10 mM MgCl2, 1 mM DTT, 5% glycerol)
in a final volume of 20 ml with addition of 2 mg of poly dI:dC, 0.5 mg
of plasmid DNA and 250 or 500 ng of the O protein. Samples were
incubated for 15 min at 30
C. Following incubation, they were
placed on ice, and 1 ml of 40% saccharose was added to stop the
reaction. Electrophoresis was performed in 0.5x TBE, 1% agarose gel
Fig. 3. Overproduction of lambdoid phage O proteins. A portion of each culture (1
OD600 unit, approximately 1  109 bacterial cells) was centrifuged and cell pellet was
resuspended in 20 ml of water and 5 ml of 4x Roti-load sample buffer (Roth). Samples
were incubated for 5 min at 95
C before loading on 12% SDS-polyacrylamide gels. Gels
were stained with colloidal Coomassie Brilliant Blue. Panel A: overproduction of lO
and O p27 proteins: lane 1 eprotein molecular weight marker; 2, 3, 6, 7 e cell extracts
before induction; 4, 5 e lO extracts after IPTG induction; 8, 9 e O P27 extracts after
induction. Panel B: overproduction of 933W O protein: lane 1 eprotein molecular
weight marker; 3, 5, 7, 9, 11 e cell extracts before induction; 4, 6, 8, 10, 12 e extracts
after IPTG induction. Black arrows indicate overproduced O proteins positions.
 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26 21

Roti-load sample buffer (Roth) was added, and samples were incu-
bated for 5 min at 95
C. Electrophoresis of cross-linked complexes
was performed in 12% SDS-polyacrylamide gel in TGS buffer. Elec-
trotransfer on a PVDF (Millipore) membrane was performed in a
Transfer buffer (0.025 M Tris, 0.192 M glycine, 20% methanol) over-
night at 20 V at 4
C. Detection of cross-linked complexes was ach-
ieved by immunoblot assay, using anti-lO polyclonal antibodies.
Visualization of antibody-antigen complexes was performed using
an ECL-Western Blotting Substrate Kit (Pierce), according to the
manufacturer's manual. For efficient O proteins' detection, we used
1:500 primary polyclonal rabbit anti-lO antibodies and 1:3000
secondary HRP-conjugated anti-rabbit antibodies. Oligomers of O
proteins were visualized using Typhoon scanner (GE Healthcare).
3. Results
3.1. Nucleotide and amino acid sequences of o genes
Nucleotide and amino acid alignments, created with use of the
Bioedit software, of three o genes sequences derived from lambdoid
bacteriophage: l, P27 and 933W, indicated differences amongst
them (Fig. 1).
There are many single-nucleotide substitutions in compared
sequences, but only two of them are translated onto amino acid
alternations. Stx-phage contain two additional binding sites for the
initiator O protein, known as iterons. Their nucleotide sequence
covers 13 additional amino acids (IPQNEGGSPKMRD). Besides them
Stx-phage O proteins have a couple of functional substitutions
compared to l phage e O protein from phage P27 has glutamic acid
instead of glycine at position 17 and isoleucine instead of leucine at
position 37 in the N-terminal domain responsible for DNA binding.
Apart from these two substitutions, phage 933W O protein revealed
another one e glycine substituted by serine at position 282 in the C-
terminal domain, taking part in interactions with another replica-
tion viral protein e P (Fig. 2). Nucleotide sequence of the o gene
contains 900 bp which corresponds to 299 amino acids of
approximately 33.9 kDa molecular weight. Stx-phage O proteins,
with additional 39 bp contain 939 bp which results in 312 amino
acid protein with molecular weight of 35.3 kDa. These findings are
in good agreement with protein electrophoretic migration in
polyacrylamide gels (Figs. 3, 4 and 6). Although Stx-phage O pro-
teins have slightly different amino acid composition than lO, their
isoelectric points are very similar e 9.18 for Stx-phage O proteins
and 9.37 for lO.

Fig. 4. Purification of O proteins on a HisTRAP HP (GE Healthcare) column. 20 ml of each fraction was collected and incubated for 5 min at 95
with addition of 5 ml of 4x Roti-load
sample buffer (Roth). Samples were electrophoresed on a 12% SDS-polyacrylamide gel and stained with colloidal Coomassie Brilliant Blue. Panel A: lO: lane 1 e protein molecular
weight standard; 2e10 e fractions containing lO protein. Panel B: P27 O: 1 e protein molecular weight standard; 2e6 e fractions containing O protein. Panel C: 933W O: 1 e
protein molecular weight standard; 2e6 e fractions containing O protein.
22 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26
Fig. 5. Chromatograms of purification of lO and O 933W proteins on a HisTRAP col-
umn. Panel A - lO protein peak 1700 mAU high occurred at 80e100% of Elution Buffer
which corresponded to about 0.4e0.5 M of imidazole. B - In case of Stx-phage, pro-
teins' peaks occurred at 50e70% of Elution Buffer, which responded to about
0.26e0.36 M of imidazole. Peak of O 933W protein was about 2200 mAU high. Black
arrows indicate pooled fractions.
3.2. Overproduction of O proteins
We overproduced studied proteins with C-terminal 6x His-tag
with the use of a powerful expression pET System. O genes were
Fig. 6. Purification of O proteins on a Superose 12 10/300 GL column (GE Healthcare). 20 ml of each fraction was incubated for 5 min at 95
with addition of 5 ml of 4x Roti-load
sample buffer (Roth). Samples were electrophoresed in a 12% SDS-polyacrylamide gel and stained with colloidal Coomassie Brilliant Blue. Panel A: lO: lane 1 e protein molecular
weight standard; 2e4 e fractions containing lO protein. Panel B: P27: 1 e protein molecular weight standard; 2e8 e fractions containing O protein. Panel C: 933W: 1 e protein
molecular weight standard; 4e7 e fractions containing O protein.
cloned into pET24a plasmid under control of the strong T7 pro-
moter. Overproduction was carried out in Escherichia coli Rosetta
(DE3) pLysS host strain at 30
C. Gene overexpression was induced
by addition of IPTG. From 1 L of the culture we obtained 3e6 g of
bacterial cell paste. Densitometric analysis of gels indicated that
putative amount of each O protein was as high as 10e20% of a total
cellular protein content (Fig. 3).
3.3. Bacterial cell lysis and ammonium sulfate fractionation
Bacterial cell lysis of 6 g of cell paste was performed according to
the method described by Roberts and MacMacken [24] with couple
of modifications. Procedures of gentle cell lysis permitted acquisi-
tion of desired quantities of studied proteins in a soluble fraction
(Fig. 3). In the case of Stx-phage O proteins, the additional presence
of nonionic detergent Triton® X-100, enhanced their recovery in a
soluble fraction. Ammonium sulfate fractionation in two steps
allowed us to concentrate and conduct initial purification of pro-
teins by removing large amounts of contaminant proteins before
loading them on a column.
3.4. Purification of O proteins
Three His-tagged O proteins derived from l and two Stx-phage:
P27 and 933W, were purified to a near homogeneity with the use of
two columns: HisTRAP and Superose 12 10/300 GL in AKTA € FPLC
system. First step of purification was performed on a HisTRAP
column (Figs. 4 and 5). It included 3 steps of washing column in an
increasing concentration of Elution buffer, and a gradient step in
which proteins were eluted. Elution of lO protein occurred at
80e100% of Elution Buffer which corresponded to about 0.4e0.5 M
of imidazole. In case of Stx-phage proteins, peaks occurred at
50e70% of Elution Buffer, which corresponded to about
0.26e0.36 M of imidazole. Densitometry analysis, conducted in
Quantity One 4.6.6. program, indicated that in this step of purifi-
cation we obtained lO protein 70%, P27 O 80% and 933W 60% pure.
Therefore another step of cleansing was needed. Imidazole and
high concentration of salt was removed by a dialysis, and a second
step of protein isolation was accomplished with a size-exclusion
chromatography on a Superose 12 10/300 GL column (Figs. 6 and
7). In case of 993W O protein, this step had to be repeated for
acquisition of homogeneous protein. O proteins' peaks in size-
exclusion chromatography occurred after eluting 11e12 ml of
buffer which corresponded to molecular weight of protein dimer e
approximately 70 kDa, according to instructions given by the col-
umn producer. Molecular weights of O proteins established by gel
 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26 23

3.5. Binding of purified O proteins to plasmids containing origins of

DNA replication

EMSA assays conducted with purified O proteins revealed that

all three of them are active and bind efficiently and specifically to
plasmids containing l or Stx-phage origins of DNA replication
(Fig. 8). All of O proteins variants did not bind plasmid pUC18
without origin of replication, specific DNA sequence. Moreover, in
case of both origins, shifts are approximately the same, thus mo-
lecular weight of the most complex structures seems to be similar.
To get more insight into these DNA-protein complexes and oligo-
merization of O proteins, DSG cross-linking assay was performed
(Fig. 9). Results of this assay showed that the functional unit in DNA
binding for each O protein is the dimer, as we can conclude from
comparing migration of DNA-protein complexes with molecular
mass marker. These results confirm previous assumption that
complexes formed by Stx-phage O proteins with both origins have
approximately the same molecular mass as l phage O protein
complex, which implies that various O proteins binds only four
iteron sequences in case of oriStx.

4. Discussion

The O protein plays an important role in lambdoid bacterio-

Fig. 7. Chromatograms of purification of lO and O 933W proteins on a Superose 12 10/
300 GL column. lO protein peak 265 mAU high (Panel A), and 933W O peak 555 mAU
high (B), occurred after eluting 11e12 ml of buffer which corresponded to molecular
weight of approximately 70 kDa according to instructions given by the column pro-
ducer and is with an agreement with molecular mass of protein O dimers. Black arrows
indicate pooled fractions.
filtration are in a good agreement with those calculated in Bioedit
program as well as with protein migration in polyacrylamide gels
(Fig. 6). All three proteins were >90% pure after executing this last
step of purification. After combining fractions with purified O
proteins and supplemented them with glycerol to a final concen-
tration of 50% we acquired proteins in following amounts and
concentrations: 7 ml of lO protein in 3.2 mg/ml solution e which
corresponds to 22.4 mg of protein in total; 10 ml of P27 O in 2.4 mg/
ml e 24.0 mg; and 7 ml of 933W O in 4.0 mg/ml e 28.0 mg.
Summarizing, we managed to obtain 22.4e28.0 mg of >90% pure
protein in a 2.4e4.0 mg/ml concentration from 6 g of bacterial cell
paste. Yields of purification steps are summarized in Table 1.
phage development as an initiator of replication of the genetic
material. Effective DNA replication is necessary for efficient pro-
duction of Shiga toxin, which is the main virulence factor of STEC
strains. It was shown that DNA replication of l bacteriophage is
dependent on the presence of the lO protein [30]. Stx-phage O
proteins were never purified before. L phage O protein was already
isolated [24,31,32] but with serious difficulties of its over-
production and purification.
Problems with overproduction and purification of lO protein in
previous studies resulted from its highly unstable nature in vivo (t1/
2 ¼ 1.5 min) and tendency to aggregate [24]. Stx-phage O proteins
have slightly different molecular weight and amino acid composi-
tion than lO. Although it does not result in significantly changed
isoelectric point, which is 9.18 for Stx’ and 9.37 for l phage O
proteins, it may considerably change their properties and confor-
mation which could result in even more serious problems with
their stability and what follows, isolation. Though the presence of a
stable fraction of O proteins in bacteria bearing plasmids derived
from Stx-phage was found [16], we encountered problems with
isolating significant quantities of these initiation factors. This could
mean that in vivo these proteins are even less stable and soluble
than lO protein.
Previously described protocols of lO protein overproduction
were based on thermoinduction systems with the use of plasmids
containing fragments of l phage origin of replication, including o
and p genes under control of pL or tandemly arrange pL and pR
promoters [33,24]. Expression of proteins in those systems was
repressed by a thermosensitive repressor and upon elevation of
temperature lO was overproduced to extent of several percent of
the total cellular proteins. These systems required lots of medium
to grow bacteria and to heat cell cultures for thermal inactivation of

Table 1
Summarized purification of O proteins. Percentage of purity in all stages and amount of O proteins in lysates were estimated by densitometry analyses.

Protein Lysate Purification on a HisTRAP column Size-exclusion chromatography

Total protein O protein Yield Purity Total protein O protein Yield Purity Total protein O protein Yield Purity
(mg) (mg) (%) (%) (mg) (mg) (%) (%) (mg) (mg) (%) (%)

lO 695 75 100 11 36 25 33 70 24 22 30 95.

P27 O 678 70 100 10 34 27 39 80 26 24 34 94.
933W O 698 80 100 12 48 29 36 59 29 28 35 97
24 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26

Fig. 8. Binding of the different O proteins to various plasmids containing l or Stx-phage origin of DNA replication. Samples with 500 ng plasmid DNA and various amounts of O
protein were electrophoresed on 1% agarose gel at 4
C, 80 V for 3.5 h. Gel was stained with 0.1 mg/ml ethidium bromide solution. Lanes 1e9 - pUC18 with: 2e250 ng of lO;
3e500 ng of lO; 5e250 ng of P27 O; 6e500 ng of P27 O; 8e250 ng of 933W O; 9e500 ng 933W O; lanes 10e18 - pUC18_oril with: 11e250 ng of lO; 12e500 ng of lO; 14e250 ng of
P27 O; 15e500 ng of P27 O; 17e250 ng of 933W O; 18e500 ng of 933W O; lines 19e27 - pUC18_oriStx with: 20e250 ng of lO; 21e500 ng of lO; 23e250 ng of P27 O; 24e500 ng of
P27 O; 26e250 ng of 933W O; 27e500 ng or 933W O. All three purified proteins revealed strong interaction with each plasmid DNA containing origin sequence.

Fig. 9. Oligomerization of purified O proteins. 20 ml of cross-linked plasmid-protein samples were incubated with 5 ml of 4x Roti-load sample buffer (Roth) for 5 min at 95
C and
electrophoresed on 12% SDS-polyacrylamide gel. Protein-protein complexes were visualized by immunoblotting with anti-lO polyclonal antibodies (panel A) or colloidal Coomassie
Brilliant Blue staining (Panel B). Lane 0 e molecular mass marker; 1 e lO; 2 e lO þ pUC18_oril; 3 e lO þ pUC18_oriStx; 4 e P27 O; 5 e P27 O þ pUC18_oril; 6 e P27
O þ pUC18_oriStx; 7e933W O; 8e933W O þ pUC18_oril; 9e933W O þ pUC18_oriStx.

the repressor. Both protocols stated that about 2.5 g of wet cell
paste were obtained from 1 l of culture. Moreover, overexpression
of the p gene was proved to be toxic to bacterial host cells [34], and
therefore it interferes with efficient overproduction of desired lO
protein. Therefore we cloned only the o gene to a high copy number
plasmid pET24a with a strong T7 promoter. Overproduction system
designed in our study turned out to be more efficient than previous
ones. We obtained 3e6 g of paste per every liter of bacterial culture.
No need of preheated media addition was also an improvement
relative to previous systems, as our cultures were grown without
changing any factors in a continuous matter, only with an addition
of IPTG. Our method allowed us to obtain a high yield of
 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26
overproduced O proteins (10e20% of total cellular proteins) in a
relatively small volume of E. coli cultures (Fig. 3). Great over-
production efficiency allowed us to isolate studied proteins in a
desirable yield and purity suitable for many molecular analyses.
We also improved bacterial cell lysis methods. We chose not to
include sonication, as it was described previously [32], to eliminate
potential aggregates. Instead, performed a simple, gentle lysis
where we exposed bacterial cells to high temperature shocks. The
lysis protocol is similar to that described by Roberts and McMacken
[24], but with addition of detergent and spermidine. The use of
detergent and high concentration of salt allowed us to keep pro-
teins in a stable, soluble state, and to remove background impu-
rities to a great extent. Since O proteins bind efficiently to DNA,
addition of spermidine was a considerable improvement of the
isolation protocol, as it effectively precipitates DNA.
We engineered O proteins with C-terminal 6x His-tag, thus, we
could purify them with the use of affinity chromatography. The C-
terminal domain of the O protein does not take part in DNA binding,
hence we could assume that addition of the Tag should not affect
results of our experiments. In our method, we used only two col-
umns, which is another upgrade of previous isolations (Figs. 4e7). For
example, Tsurimoto et al. [31] used a time consuming, four column
system. As a result of our actions, we managed to obtain 22.4e28.0 g
of >90% pure protein from 6 g of bacterial cell paste, which is suitable
for molecular investigations. Our protocol is easy to up-scale, only by
changing the column volume. We tested HisTRAP column with
higher volume (5 ml) and obtained greater amount of purified pro-
tein. Nevertheless for our requirements 1 ml column was enough.
With the use of an EMSA assay and DSG cross-linking assay, we
established that all three isolated proteins were active in DNA
binding and that these interactions are specific e O proteins bound
only to DNA containing phage origin sequences (Figs. 8 and 9).
Moreover, every protein interacted with DNA as a dimer, thus we
conclude that the functional units of l and Stx phage O proteins are
dimers. Furthermore, shifts in DNA-protein complexes migration in
conducted assays were approximately the same in case of both
origins, therefore, molecular weights of the complexes seem to be
similar. This may suggest that not all of six iterons present in Stx-
phage origin of replication are occupied by proteins, therefore,
the complex is smaller than expected. One may hypothesize that
various O proteins bind only four iteron sequences in case of oriStx,
hence only four protein dimers, instead of six form Stx-phage “O-
some” complexes, like in l phage. However, this supposition must
be verified by methods of molecular biology, which would give a
better insight into “O-some” complex formation and mode and
order of iteron binding by different proteins.
5. Conclusion
Improved method for overexpression of l o gene and purifica-
tion of its protein has been developed. Using this method the O
proteins of Shiga toxin-converting phage P27 and 933W were pu-
rified for the first time. O proteins of phage l, P27 and 933W have
been demonstrated to be active in binding to origin regions of
bacteriophage l and P27 genomes.
Acknowledgements
This work has been supported by The National Science Center
(NCN), government of Poland (grant No UMO/2011/02/A/NZI/
00009).
References
[1] D.L. Hartl, D.E. Dykhuizen, The population genetics of Escherichia coli, Annu.
25
Rev. Genet. 18 (1984) 31e68.
[2] R.E. Besser, P.M. Griffin, L. Slutsker, Escherichia coli O157:H7 gastroenteritis
and the hemolytic uremic syndrome: an emerging infectious disease, Annu.
Rev. Med. 50 (1999) 355e367.
[3] C.L. Gyles, Shiga toxin-producing Escherichia coli: an overview, J. Anim. Sci. 85
(13) (2007) E45eE62.
[4] A.T. Serna, E.C. Boedeker, Pathogenesis and treatment of Shiga toxin-
producing Escherichia coli infections, Curr. Opin. Gastroenterol. 24 (1) (2008)
38e47.
[5] D. Law, Virulence factors of Escherichia coli O157 and other Shiga toxin-
producing E. coli, J. Appl. Microbiol. 88 (5) (2000) 729e745.
[6] J.M. Los, M. Los, G. Wegrzyn, Bacteriophages carrying Shiga toxin genes:
genomic variations, detection and potential treatment of pathogenic bacteria,
Future Microbiol. 6 (8) (2011) 909e924.
[7] S.K. Bloch, A. Felczykowska, B. Nejman-Falenczyk, Escherichia coli O104:H4
outbreakehave we learnt a lesson from it? Acta Biochim. Pol. 59 (4) (2012)
483e488.
[8] H. Karch, E. Denamur, U. Dobrindt, B.B. Finlay, R. Hengge, L. Johannes, E.Z. Ron,
T. Tønjum, P.J. Sansonetti, M. Vicente, The enemy within us: lessons from the
2011 European Escherichia coli O104:H4 outbreak, EMBO Mol. Med. 4 (9)
(2012) 841e848.
[9] B. Nejman, J.M. Los, M. Los, G. Wegrzyn, A. Wegrzyn, Plasmids derived from
lambdoid bacteriophages as models for studying replication of mobile genetic
elements responsible for the production of Shiga toxins by pathogenic
Escherichia coli strains, J. Mol. Microbiol. Biotechnol. 17 (4) (2009) 211e220.
[10] K. Taylor, G. Wegrzyn, Replication of coliphage lambda DNA, FEMS Microbiol.
Rev. 17 (1e2) (1995) 109e119.
[11] C. Alfano, R. McMacken, Heat shock protein-mediated disassembly of nucle-
oprotein structures is required for the initiation of bacteriophage lambda DNA
replication, J. Biol. Chem. 264 (18) (1989) 10709e10718.
[12] K. Zahn, F.R. Blattner, Binding and bending of the lambda replication origin by
the phage O protein, EMBO J. 4 (13A) (1985) 3605e3616.
[13] T. Tsurimoto, K. Matsubara, Purified bacteriophage lambda O protein binds to
four repeating sequences at the lambda replication origin, Nucleic Acids Res. 9
(8) (1981) 1789e1799.
[14] J.B. Mallory, C. Alfano, R. McMacken, Host virus interactions in the initiation of
bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB
helicase by lambda P replication protein, J. Biol. Chem. 265 (22) (1990)
13297e13307.
[15] M. Narajczyk, S. Baran
ska, A. Wegrzyn, G. Wegrzyn, Switch from theta to
sigma replication of bacteriophage lambda DNA: factors involved in the
process and a model for its regulation, Mol. Genet. Genomics 278 (1) (2007)
65e74.
[16] B. Nejman, B. Nadratowska-Wesolowska, A. Szalewska-Palasz, A. Wegrzyn,
G. Wegrzyn, Replication of plasmids derived from Shiga toxin-converting
bacteriophages in starved Escherichia coli, Microbiology 157 (Pt 1) (2011)
220e233.
[17] K. Mensa-Wilmot, K. Carroll, R. McMacken, Transcriptional activation of
bacteriophage lambda DNA replication in vitro: regulatory role of histone-like
protein HU of Escherichia coli, EMBO J. 8 (8) (1989) 2393e2402.
[18] A. Szambowska, M. Pierechod, G. Wegrzyn, M. Glinkowska, Coupling of
transcription and replication machineries in lambda DNA replication initia-
tion: evidence for direct interaction of Escherichia coli RNA polymerase and
the lambda O protein, Nucleic Acids Res. 39 (1) (2011) 168e177.
[19] B. Lipinska, A. Podhajska, K. Taylor, Synthesis and decay of lambda DNA
replication proteins in minicells, Biochem. Biophys. Res. Commun. 92 (1)
(1980) 120e126.
[20] D. Wojtkowiak, C. Georgopoulos, M. Zylicz, Isolation and characterization of
ClpX, a new ATP-dependent specificity component of the Clp protease of
Escherichia coli, J. Biol. Chem. 268 (30) (1993) 22609e22617.
[21] A. Wawrzynow, D. Wojtkowiak, J. Marszalek, B. Banecki, M. Jonsen, B. Graves,
C. Georgopoulos, M. Zylicz, The ClpX heat-shock protein of Escherichia coli, the
ATP-dependent substrate specificity component of the ClpP-ClpX protease, is
a novel molecular chaperone, EMBO J. 14 (9) (1995) 1867e1877.
[22] A. Wegrzyn, A. Czyz, M. Gabig, G. Wegrzyn, ClpP/ClpX-mediated degradation
of the bacteriophage lambda O protein and regulation of lambda phage and
lambda plasmid replication, Arch. Microbiol. 174 (1e2) (2000) 89e96.
[23] M. Zylicz, D. Ang, K. Liberek, T. Yamamoto, C. Georgopoulos, Initiation of
lambda DNA replication reconstituted with purified lambda and Escherichia
coli replication proteins, Biochim. Biophys. Acta 951 (2e3) (1988) 344e350.
[24] J.D. Roberts, R. McMacken, The bacteriophage lambda O replication protein:
isolation and characterization of the amplified initiator, Nucleic Acids Res. 11
(21) (1983) 7435e7452.
[25] R.C. Mierendorf, B.B. Morris, B. Hammer, R.E. Novy, Expression and purifica-
tion of recombinant proteins using the pET system, Methods Mol. Med. 13
(1998) 257e292.
[26] C. Yanisch-Perron, J. Vieira, J. Messing, Improved M13 phage cloning vectors
and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors,
Gene 33 (1) (1985) 103e119.
[27] A. Wegrzyn, G. Wegrzyn, K. Taylor, Protection of coliphage lambda O initiator
protein from proteolysis in the assembly of the replication complex in vivo,
Virology 207 (1) (1995) 179e184.
[28] J. Sambrook, D.W. Russell, Molecular Cloning: a Laboratory Manual, third ed.,
Cold Spring Harbor Laboratory Press, New York, 2001.
[29] N. Dyballa, S. Metzger, Fast and sensitive colloidal coomassie G-250 staining
26 K.I. Kozłowska et al. / Protein Expression and Purification 131 (2017) 16e26
for proteins in polyacrylamide gels, J. Vis. Exp. 30 (2009).
[30] M. Zylicz, D. Ang, K. Liberek, C. Georgopoulos, Initiation of lambda DNA
replication with purified host- and bacteriophage-encoded proteins: the role
of the dnaK, dnaJ and grpE heat shock proteins, EMBO J. 8 (5) (1989)
1601e1608.
[31] T. Tsurimoto, K. Matsubara, Purification of bacteriophage lambda O protein
that specifically binds to the origin of replication, Mol. Gen. Genet. 181 (3)
(1981) 325e331.
[32] M. Zylicz, I. Gorska, K. Taylor, C. Georgopoulos, Bacteriophage lambda
replication proteins: formation of a mixed oligomer and binding to the origin
of lambda DNA, Mol. Gen. Genet. 196 (3) (1984) 401e406.
[33] T. Tsurimoto, T. Hase, H. Matsubara, K. Matsubara, Bacteriophage lambda
initiators: preparation from a strain that overproduces the O and P proteins,
Mol. Gen. Genet. 187 (1) (1982) 79e86.
[34] S. Hayes, C. Erker, M.A. Horbay, K. Marciniuk, W. Wang, C. Hayes, Phage
Lambda P protein: trans-activation, inhibition phenotypes and their sup-
pression, Viruses 5 (2) (2013) 619e653.