The Study of Colicin in the Limited Protection of the Colicin Producing Cells
against Bacteriophage M13
Yuh-Ren Chen, Chen-Chung Liao, Kin-Fu Chak
Institute of Biochemistry and Molecular Biology, National Yang Ming University,
Shih-Pai, Taipei 11221, Taiwan, ROC
Abstract
Colicin is a plasmid encoded bacteriocin which is produced by and active against
Escherichia coli and closely related bacteria, thus the plasmid is known as Col
plasmid. Recently it has been reported that E. coli cells harboring the Col plasmid
could confer limited protection against bacteriophage infection, which may be one of
the reasons for the coexistence of E. coli with the Col plasmid. We therefore further
investigate which functional domain of ColE7 exerts the protective effect against
bacteriophage M13. Subsequently, we found that only the translocation and receptor
binding domain (TR domain) conferred limited protection against M13 phage
infection. In contrast to the protective effect of TR domain against M13 infection, the
endogenous expressed TR domain did not confer limited protection against
transfection of the M13 phage DNA. Probably, this observation indicated that the
protective effect may occur in the early stage during phage DNA injection through the
cell membrane. Using BIAcore biosensor, we have found that the TR domain could
interact with M13 phage particle in vitro. These results have been further confirmed
by in vivo cross-linking that TR protein could interact with major coat proteins, g8p. It
was known that g8p is required for the phage infection and also involved in the
insertion into the cytoplasmic membrane. Base on these experimental results of
protein-protein interactions, we would like to propose that overexpression of TR
domain might interact with g8p during infection, leading to the interference of g8p
integrating into the membrane, thus cause the cells conferring limited protection
against M13 phage infection as a result.
Colicins are bactericidal protein toxins which are active against Escherichia coli
and its related strains of E. coli [1,2]. They are encoded by colicinogenic plasmids
(pCol) which are widely distributed in natural populations of E. coli. Colicin serve
some function in microbial population without dispute, but the ecological role of
colicin is still inconclusive [3]. Recently, our group indicated that E. coli containing
ColE7-K317 can confer limited protection against bacteriophage M13K07 and λ
infection [4]. This observation is consistent with the suggestion that the phenomenon
of limited bacteriophage resistance might play an important role in maintaining the
high-level population of plasmid-containing cells in natural environment [5]. Based
on this observation, we propose that the pathway of colicin secretion might encounter
to some extent with the bacteriophage infection pathway leading to interference of
bacteriophage infection. The limited protection conferred by colicin producing cells
might provide a different defense mechanism other than well-known
restriction/modification system, resistance induction, superinfection exclusion, and
abortive infection [6-9].
Colicins are released into the environment by colicin lysis protein, and to date
the mechanism of colicin secretion is not clear. Lysis gene is the last gene in the
colicin operon, and inactive lysis protein will result in cytosolic accumulation of
colicin proteins [10,11]. The extracellular secretory mechanism of colicin is different
from that of the five known secretion pathways in gram-negative bacteria [12,13]. The
pathway of colicin secretion has been considered to be a particular system of protein
secretion in E. coli. It is indicated that colicin A produced in the cytoplasm can
translocate to periplasm in the presence of lysis protein, and the presence of colicin A
in the periplasm might also interact with the Tol proteins which would result in
nonfunctional Tol proteins and tol phenotype [14,15]. The phenomenon of the
producing cells is similar to that of tol mutants [16]. It is suggested that colicin
exportation is similar to colicin import [17].
M13 bacteriophage is one of the members of filamentous bacteriophages, which
only adsorbs on F＋ E. coli cells by interacting with F conjugative pilus [18]. This
interaction is initiated by the specific binding of the central domain (N2) of the minor
coat protein g3p to the tip of F pilus. After retraction of F pilus, the N1 domain of g3p
binds to the C-terminal domain of TolA protein. Consequently, TolA protein can bring
the outer and inner membranes of the bacteria closer together facilitating the transport
of the phage DNA into the cytoplasm of a host cell [19,20]. However, phage DNA
transportation during infection is also accompanied by insertion of the major coat
protein, g8p, into the cytoplasmic membrane [21]. Further, functioning TolQ, TolR,
and TolA proteins are required for insertion of g8p into cytoplasmic membrane upon
infection [22]. This finding indicates that both colicin import and phage infection
need the assistance of Tol protein system.
In this work, we found that only the translocation and receptor binding domain
(TR domain) conferred limited protection against M13 phage infection. The
protective effect may occur in the early stage during phage DNA injection through the
cell membrane. Moreover, it was indicated that TR protein could interact with major
coat proteins, g8p. Thus, this result indicates that TR domain might interfere with the
insertion of g8p into the cytoplasmic membrane, causing the limited protection
against M13 phage infection.
Materials and methods
Bacterial strains, media, and construction of plasmids. E. coli JM109 F′ was
used as the host strain for subcloning, expression, and bacteriophage infection. The
expression plasmids pQE30, pQE70 (Qiagen) were used for the over-expression of
the T, R, TR, T2A-Im, Im-lys domains. M13K07 bacteriophages were purchased from
Institute of Food Industry Research and Development, Shin-Chu, Taiwan. Cultures
were routinely grown in Luria–Bertani (LB) broth or on plates of LB agar and were
supplemented, where required, with ampicillin (100 μg ml−1).
Construction of a vector for expression of the individual functional domains. The
cloning method was described in Lin et al. [4]. A DNA fragment containing the
translocation domain and receptor binding domain of the ColE7 operon was amplified
by PCR using a pair of oligonucleotide primers:
Col7-701 (5’-GAATTTGCATGCGCGGTGG-3’) and TR-702
(5’-GACAGGAGATCTTTTA CCTGT-3’).
The translocation domain: Col7-701 (5’-GAATTTGCATGCGCGGTGG-3’) and
T-702 (5’-GCGTTAAGCAGATCTCACCGG-3’).
The receptor binding domain: R-701 (5’-CCGGTGGGCATGCCTGAACGCAAT)
and TR-702 (5’-GACAGGAGATCTTTTA CCTGT-3’).
The T2A-Im complex: T2A701 (5’-GCTAAAGGCATGCTAGATAAGG) and Im302
(5’-CATAAGCTTTCCTTGTTGTGAAAGAAT-3’).
The immunity protein: Im301 (5’TAATATGGGATCCAAAAATAGTATTAGTG-3’)
and Im302 (5’-CATAAGCTTTCCTTGTTGTGAAAGAAT-3’).
The lysis protein: ImF (5’-AATAATAGCATGCTGAAAAATAG-3’) and LysR
(5’-GGATTGCAGATCTCTGCGTTTC-3’)
Infection experiments. Bacteria were grown in LB broth at 37 °C to an
absorbance at 600 nm of 0.6. The expression of protein was induced with 1 mM IPTG
for 1.5 h. Thereafter, phage particles were added to the bacterial cells at multiplication
of infection (MOI) of 0.01 and allowed to infect for 15 min at 37 °C. The samples
were plated in soft agar on LB plates. The plates were incubated overnight at 37 °C.
The number of plaques forming units (PFU) for each of the bacteria was determined
by counting the PFU on each plate. The experiments were performed in triplicate.
Transfection of phage ssDNA by heat shock. Bacterial cells containing
pQE30-TR were grown in LB broth at 37 °C to an absorbance at 600 nm of 0.4. The
expression of TR domain was induced with 0.1 mM IPTG for 30 min. The bacterial
cells were harvested and competent cells were prepared by calcium chloride method
(Molecular Cloning, Cold Spring Harbor Laboratory Press). The phage ssDNA were
isolated from M13 phage particles by QIAprep® Spin M13 Kit (Qiagen). The isolated
M13 phage ssDNA were transformed into the endogenous expressed TR domain
competent cells. The samples were plated in soft agar on LB plates. The plates were
incubated overnight at 37 °C. The number of plaques forming units (PFU) for each of
the bacteria was determined by counting the PFU on each plate. The experiments
were performed in triplicate. The respective protein expression level was determined
by Western blotting by using polyclonal antibodies raised against his-tag as described
in Lin et al. [4].
Purification of TR, T2A-Im, Im domain proteins. Overnight cultures (10 ml) of
JM109 (pQE30-TR, pQE70-T2A-Im, pQE30-Im) were diluted into 1 L LB and
incubated with shaking at 37 °C for 4–5 h. IPTG was added to a final concentration of
1 mM to induce the expression of the respective domains for 3 h. Cells were harvested
and disrupted by French pressure. Ni–NTA resin affinity column (Qiagen, Germany)
was equilibrated with sonication buffer (50 mM sodium phosphate buffer, pH 7.8, and
300 mM NaCl) before being loaded with the crude cell extracts (40 ml). After loading
and binding with Ni-NTA resin, the column was washed with 6 bed volumes (in terms
of packed resin volume) of washing buffer (50 mM sodium phosphate buffer, pH 7.0,
300 mM NaCl, and 75 mM imidazole) to remove the unbound protein. The bound
protein was then eluted by 4 bed volumes of elution buffer (50 mM sodium phosphate
buffer, pH 7.0, 300 mM NaCl, and 500 mM imidazole). The purity of respective
proteins are analyzed by SDS-PAGE. The proteins are concentrated and dialyzed
against phosphate buffer saline (sigma) and stored at -20℃ for in vitro interaction
assay.
Analysis of protein-M13 phage particles interaction by surface plasmon resonance.
The interaction between individual domain and M13 phage particle was measured by
SPR using the BIAcore X system. Typical SPR experiment had the following stpes：(1)
The NTA sensor chip was first equilibrated with running buffer (20 mM sodium
phosphate, 150 mM NaCl, pH 7.0) and then regenerated with injections of 40 μl
regeneration buffer (500 mM EDTA). (2) NTA chip surfaces were charged with
nickel ions by passing 40 μl of 0.5 mM NiSO4. (3)The purified TR domain, Immunity
protein or T2A-Im complex was individually bound via their hexahistidine tags to the
nickel surface in flow cell 1. (4) A further brief wash with wash buffer (20 mM
sodium phosphate, 150 mM NaCl, pH 7.0) was applied to release some ligands of low
binding affinity from the surface. (5) The concentrated bacteriophage M13K07
particles was analyzed by passing from flow cell1 to flow cell 2. After the analysis of
each time, the step was repeated from step (1). All SPR experiments within the
BIACORE instruments were carried out at 25 °C, and the flow rate is 20 μl min−1.
The binding curves were measured in RU, and the affinity of association and
dissociation was analyzed by BIAevaluation.
In vivo crosslinking and pull-down by Ni-NTA beads. E. coli cells infected by
bacteriophage M13K07 for 10 min were subjected to be cross-linked for 30 min at
room temperature with 1% formaldehyde. After cross-linking, the reaction was
quenched by 125 mM Tris-HCl for 20 mins. Then, the cell pellet was resuspended by
sonication buffer (20 mM Tris-HCl, 100 mM Na2HPO4, 0.2% SDS, 0.1% Tween-20,
8M urea, pH 7.8) and lysed by French pressure. The cell lysates were incubated with
pre-equilibrated Ni-NTA resin for 1 hr and the TR associated proteins were pulled
down. After washed by wash bufferI (20 mM Tris-HCl, 100 mM Na2HPO4, 0.1% SDS,
0.1% Tween-20, 8M urea, 50mM imidazole, pH 7.0)to remove non-specifically
binding proteins, the beads were further washed by wash bufferII (50 mM sodium
phosphate buffer, 300 mM NaCl , pH 7.0) to remove the residual detergent. The TR
associated complex was eluted by elution buffer. The eluate was mixed with 4X
sample buffer and boiled at 100℃ for 20 mins. Then, it was analyzed by western
blotting.
Results
Limited protection against bacteriophage M13K07 was conferred by TR domain
Using number of plaque-forming units as a standard to categorize the limited
protection of E. coli cells against bacteriophage, we had previously shown that
cea-ceiE7 conferred the limited protection against bacteriophage M13K07 and it was
directly related to the induction level of the cea-ceiE7 genes [4]. Therefore, we further
investigate which functional domain of ColE7 exerts the protective effect against
bacteriophage M13. Firstly, the cea-ceiE7 genes were divided into three functional
domain, transloction and receptor binding domain (TR domain), toxicity domain and
immunity protein (T2A-Im) and immunity protein alone (Im) (Fig. 1). In the same as
previous method, expression of each individual domain is directly under the control of
the lac promoter and his-tag was fused to each domain. Thus, the expression vector
can be used to test the role of each domain in the limited protection.
The study of IPTG induction (1 mM) of E. coli JM109 (TR, T2A-Im, Im) clearly
showed that only the TR domain conferred limited protection against bacteriophage
M13K07 and it was correlated to the expression level of the TR domain. However, the
toxicity domain and immunity protein did not possess this protective ability. The
plaque-forming units were reduced to 50% compared to the non-induced cells while
the other two domain were not changed (Fig. 2). It is worth noting that the control
group (vector alone) had no effect on plaque-forming units. The expression levels of
each protein were verified by Western blotting analysis of the cell extracts.
Both T domain and R domain conferred the limited protection against
bacteriophage M13K07
Since TR domain is composed of T domain and R domain, we had further
examined which domain is involved in the limited protection. T domain mediates the
translocation of the cytotoxic domain by coordinating with the Tol proteins, while R
domain is responsible for recognition of a specific receptor, BtuB, the vitamin B12
receptor [23]. The plaque-forming units of the cells containing T domain was reduced
to 45% compared to the non-induced cells while 54% in cells containing R domain
(Fig. 3). Combined with the results of expression levels of each protein, it was
suggested that both T domain and R domain could confer the limited protection
against bacteriophage M13K07. Although the level of reduced PFU is different, it
may be due to the different protein amount by IPTG induction. Thus far, these results
suggest that TR domain plays an important role in the limited protection against
bacteriophage. However, we do not know whether the way T domain impeded the
bacteriophage is the same as that in R domain.
The lysis protein had no protective effect against bacteriophage M13K07
The lysis protein is essential for colicin secretion and high levels of lysis protein
will result in quasilysis [24]. It was not known that whether the effect of lysis protein
could influence the bacteriophage infection. In order to examine the act of lysis
protein before and after quasilysis, infection of cells was performed at 1 h and 2h
respectively after IPTG induction. Before quasilysis the lysis protein might function
in the inner membrane and it can activate OmpLA, outer membrane phospholipase A,
which can increase permeability of cell membrane. The PFU was not altered after 1 h
of IPTG induction (Fig. 4). The integrity of cell membrane was seriously affected
after quasilysis but the PFU was not significantly reduced after 2 h of IPTG induction
(Fig. 4). It cannot be ruled out that the minor effect of PFU might be due to the
destruction of cell membrane. These results also indicated that the manner of the lysis
protein in the cell membrane did not impede the bacteriophage infection and the
higher permeability of the cell membrane did not assist in the release of bacteriophage
particles. Thus far, these events point out the TR domain chiefly exerts the limited
protection against bacteriophage M13K07.
Stage of occurrence of the limited protection against bacteriophage M13K07
Filamentous M13K07 bacteriophage can infect E. coli cells and transport of
ssDNA into the cytoplasm is achieved by the aid of the Tol proteins [20,22]. By the
bacteriophage infection method as described above, we can not realize when the
limited protection occurred. It is interesting to know whether the limited protection is
mediated by the interference of transport of phage DNA or assembly of phage
particles. We transformed M13K07 bacteriophage ssDNA into E. coli cells containing
endogenous TR protein to mimic the transfection of E. coli cells without using
bacteriophage particles. The plaque-forming units were not dramatically changed as
compared to the cells with lower levels of endogenous TR protein (Fig. 5). This result
indicated that the protective effect may occur in the early stage during phage DNA
injection through the cell membrane.
The interaction between the TR domain and major coat protein, g8p
The TR domain could intrude the infection of bacteriophage M13K07 and it may
take place earlier than the complete transport of phage DNA. However, the
mechanism by which the TR domain interferes with the infection of M13K07
bacteriophage has not been elucidated clearly. We had previously shown that colicin
may confer the limited protection by interacting with phage DNA [4], but it cannot be
excluded that colicin may interact with the coat proteins of M13K07 bacteriophage
and contribute to the limited protection. In order to test the possibility, we examine
whether the TR domain could interact with M13K07 bacteriophage particles by using
surface plasmon resonance biosensor [25]. In this experiment, the purified TR domain,
immunity protein, and T2A-Im complex will be separately immobilized on NTA
sensor chip by 6 X histidine-tag, followed by injection of isolated M13K07
bacteriophage particles. The TR domain could interact with M13K07 bacteriophage
particles, which is observed by the binding curve, while the T2A-Im complex and
immunity protein could not as no response was detected on sensorgram (Fig. 6).
It was known that the minor coat protein, g3p, is essential for recognition of
F-pilus and penetration of bacteriophage and proper membrane insertion of the major
coat protein, g8p, is related to the translocation of bacteriophage DNA. Therefore, we
examine whether the TR domain could interact with phage coat proteins to interfere
with the infection of E. coli cells. We performed in vivo crosslinking experiments to
examine whether the TR domain could interact with the major coat protein, g8p, in E.
coli cells. E. coli cells infected with M13 K07 bacteriophage were treated in vivo with
formaldehyde, a reversible short-range (2 Å) crosslinker [26]. After crosslinking, E.
coli cells were lysed and the TR domain-associated complex was pulled down by
Ni-NTA beads and washed under denatured condition. Then, the eluates were
analyzed by Western blotting. It was found that the major coat proteins, g8p, but not
the minor coat proteins, g3p, could associate with the TR domain (Fig. 7). This result
was consistent with that of in vitro binding assay by using BIAcore biosensor.
Discussion
Colicin is a secretory protein but the mechanism of colicin secretion is not
clearly defined. Recently, we had reported that interaction between colicin and the
DNA of bacteriophage M13K07 may bring out the limited protection of
colicin-producing cells against bacteriophage [4]. Here we showed that the TR
domain contributed mostly to the limited protection against bacteriophage M13K07.
In addition to the interaction between colicin and phage DNA, the TR domain can
interact with g8p, major coat proteins, in bacteriophage M13K07 infected cells. These
results demonstrated the colicin can impede the infection of bacteriophage by
interacting with not only the phage DNA but also the phage coat proteins.
The infection of E. coli cells by bacteriophage M13K07 is a complicated process.
It was considered that the infection process is initiated by the achievement of minor
coat protein, g3p. However, the function of the major coat protein, g8p, during the
infection process is not clarified clearly. The observation that the limited protection
resulted from the TR domain interacting with g8p suggested that major coat protein,
g8p, play important roles in phage infection. Recently, Stopar et al. [27] reported that
the protein-protein and protein-lipid interaction were involved in the infection of E.
coli by bacteriophage M13. Some amino acid residues are implicated in the
membrane insertion into lipid bilayer of g8p and mutations in certain positions of the
N-terminu of g8p would reduce phage infectivity [28,29 ]. The proper insertion of g8p
for phage disassembly is followed by the transfer of phage DNA so the delicate
process needs to be correctly carried out. It was suggested that the TR domain might
interact with g8p in the inner membrane of E. coli cells and interfere the rapid
solubilisation process of g8p during phage disassembly. In our study, we showed that
colicin accounted for the limited protection by interacting with not only the phage
DNA but also the phage major coat proteins.
It is worthy to note that the TR domain could be crosslinked to the inner
membrane protein, g8p and this finding indicated that the TR domain is located near
the inner membrane. Our preliminary result also indicated that the TR domain was
located near the inner membrane by immunogold TEM (data not shown). Although
the detail mechanism of colicin release was not elucidated, it was considered that the
mechanism is different from all secretion systems in gram-negative bacteria. It is
interesting to examine whether the localization of colicin by unidentified internal
signal domain could assist the secretion of colicin to extracellular environment. On
the other hand, the lysis protein could not confer the limited protection, but it cannot
be ruled out that the lysis protein could enhance the limited protection by interacting
with colicin. Since the lysis protein of colicin A can help colicin A across inner
membrane [14], we would like to examine whether co-expression of the TR domain
and the lysis protein in cells result in reinforced limited protection. Moreover, the
localization of colicin in the cells might shed light on the mechanism of colicin
release.
It was previously reported that the efficient membrane insertion of g8p depends
on functional TolQ, TolR, and TolA proteins, which are integral cytoplasmic
membrane proteins. The translocation domain of colicin possesses the binding domain
of these Tol proteins. In addition, the TolA protein is the coreceptor of g3p and helps
the entry of phage particles. The proximity of the TR domain to the inner membrane
might provide the chance of interacting with these Tol proteins. However, the
translocation domain could result in tol phenotype only when it was fused to signal
peptide. The possibility that the TR domain might interact with the Tol proteins to
affect the function of g3p or g8p needs to be investigated further. We do not know
how the TR domain localizes near the inner membrane to interfere the infection
process of bacteriophage M13K07. Nevertheless, we would like to suggest that the
proximity of the TR domain to the inner membrane may be a cause to the limited
protection by interaction with the phage DNA and major coat protein, g8p, also, it
may help for the further pursuit of the molecular basis of colicin secretion.
FIGURE LEGENDS
cea
SOS box
cei
pColE7 cel
T
R
T2A
Imm
Fig.1. Organization of the colicin operon. ColE7-K317 plasmid contains three open
reading frames, which were under the control of SOS promoter. cea is the structural
gene of colicin and it comprises three functional domain：(i) The N-terminus is
translocation domain (T), (ii) The middle is receptor binding domain (R) (iii) The
C-terminus is cytotoxic DNase domain (T2A). cei encodes the immunity protein,
which can strongly associate with DNase domain to antagonize its enzyme activity.
cel encodes the lysis protein, which helps the release of colicin into the medium.
A
B
TR
IPTG
－
Imm
＋
－
＋
T2A-Im
－
＋
55
40
35
25
His probe
15
10
BtuB
Fig. 2. Limited protection of E. coli cells containing the different domains of colicin
against bacteriophage M13K07. (A) Total PFU of the non-induced cells in respective
domains was expressed as 100%. The PFU of the IPTG-induced cells was expressed
as percentage with respect to the total number of PFU of the cells without IPTG
induction. The experiments were performed in triplicates, and the number of
plaque-forming units (PFU) of each was shown in mean ± SD of one experiment
performed in triplicates. (B) Western blots were performed to examine the expression
levels of each domain by detecting the fused His-tag on each protein. The BtuB levels
were indicated as internal control.
A
B
TR
IPTG
－
R
T
＋
－
＋
－
＋
55
40
35
His probe
25
15
BtuB
Fig. 3. Limited protection of E. coli cells containing individual T domain or R domain
against bacteriophage M13K07. (A) Total PFU of the non-induced cells in respective
domains was expressed as 100%. The PFU of the IPTG-induced cells was expressed
as percentage with respect to the total number of PFU of the cells without IPTG
induction. The experiments were performed in triplicates, and the number of
plaque-forming units (PFU) of each was shown in mean ± SD of one experiment
performed in triplicates. (B) Western blots were performed to examine the expression
levels of each domain by detecting the fused His-tag on each protein. The BtuB levels
were indicated as internal control.
A
B
Lysis
IPTG
－
＋
His probe
BtuB
Fig. 4. Limited protection of E. coli cells containing elevated lysis protein against
bacteriophage M13K07. (A) Total PFU of the non-induced cells in respective domains
was expressed as 100%. The PFU of the IPTG-induced cells was expressed as
percentage with respect to the total number of PFU of the cells without IPTG
induction. The experiments were performed in triplicates, and the number of
plaque-forming units (PFU) of each sample was shown in mean ± SD of one
experiment performed in triplicates. (B) Western blots were performed to examine the
expression levels of the lysis protein by detecting the fused His-tag on each protein.
The BtuB levels were indicated as internal control.
A
B
TR
IPTG
－
＋
His probe
BtuB
Fig. 5. The effect of limited protection by transfection of bacteriophage M13K07
ssDNA into the E. coli cells containing endogenous TR domain. (A) The PFU of the E.
coli cells with endogenous elevated TR domain level were shown as percentage with
respect to the total number of PFU of non-induced cells. (B) The endogenous TR
domain levels were detected by Western blot. The BtuB levels were indicated as
internal control.
A
2500
Resp. Diff. (RU)
2000
1500
1000
500
0
0
50
100
150
200
250
200
250
200
250
Time (s)
B
100
Resp. Diff. (RU)
80
60
40
20
0
0
50
100
150
Time (s)
C
100
Resp. Diff. (RU)
80
60
40
20
0
0
50
100
150
Time (s)
Fig. 6. The sensorgrams for TR domain, Immunity protein, and T2A-Im complex
analyzed by interacting with bacteriophage M13K07 particles. (A) the binding curve
of TR domain and phage particles, (B) the binding curve of the Immunity protein and
phage particles, (C) the binding curves of the T2A-Im complex and phage particles.
30 TR TR TR
M13 + - + +
Formaldehyde + + - + M13
His-probe
g8p
1
2
3
4
5
Fig. 7. In vivo cross-linking of TR domain in phage-infected E. coli cells. The strains,
phage infection, formaldehyde was indicated. E. coli cells with or without
overexpressed TR domain infected with bacteriophage M13K07 was incubated with
formaldehyde for 30 min. After pull-down by Ni-NTA beads under denature condition,
the eluates were analyzed by Western blot with anti-his-tag and anti-g8p antibody.
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