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SelectiveinfectionofE.coliasafunctionofa
specificmolecularinteraction
ArticleinJournalofMolecularRecognition·January2002
DOI:10.1002/jmr.557·Source:PubMed
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JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2002; 15: 27–32
DOI:10.1002/jmr.557
Selective infection of E. coli as a function of a
specific molecular interaction
Nina Nilsson1, Fredrik Karlsson1, Jasna Rakonjac2† and Carl A. K. Borrebaeck1*
1
Department of Immunotechnology, Lund University, Lund, Sweden
Rockefeller University, New York, USA
2
Selective infection of phage is when the bacterial infection depends on the specific molecular interaction
between an antigen and a phage-displayed protein sequence such as an antibody. Engineering of the normal
infection into pathways, directed by a specific protein–protein interaction, has raised several mechanistic
questions. Here, we address the type of display and the affinity between the interacting pairs. The deleted
phage R408d3 was used for the first time in selective infection and was shown to exhibit a superior
performance compared to the VCSM13 phage. Furthermore, the affinity between the interacting pairs also
affected the selective infection process and a correlation between affinity and infection efficiency was
detected, thus implying that selective infection is the method of choice for selection of rare high-affinity
interactions in molecular libraries. Copyright # 2002 John Wiley & Sons, Ltd.
Keywords: molecular libraries; phage display; affinity of interacting pairs; R408d3; g3p
INTRODUCTION
Molecular libraries have come to play an important role in
the generation of specific antibodies and peptides to target
molecules (Vaughan et al., 1996; Aujume et al., 1997;
Hoogenboom et al., 1998; Nishikawa, et al., 2000; Wu and
Lin, 2001), and for their potential to facilitate the rapid
identification of novel gene products and new interactions in
complex networks (Borrebaeck, 1998; Pelletier et al., 1999;
Holt, et al., 2000). The filamentous phage is the most
common display vehicle utilized for molecular libraries and
Smith (1985) first demonstrated its versatility. Antigens or
antibodies have subsequently been mostly displayed on the
minor coat protein 3 (g3p), which is directly involved in the
phage infection of Escherichia coli (E. coli). However, the
display does not involve all the copies of g3p and consequently the phage particle remains infective, which makes it
possible to replicate a selected phage. Since phage libraries
can contain many billions of molecular members, one of the
challenges is to minimize non-specific binding and to enrich
the few phage particles displaying high-affinity binders over
the highly abundant low-affinity binders (Hawkins et al.,
1992). This is particularly important when using this
approach in functional genomics or in vitro evolution,
*Correspondence to: C. A. K. Borrebaeck, Department of Immunotechnology,
Lund University, PO Box 7031, S-221 07 Lund, Sweden.
E-mail: [email protected]
†BioSciences, Massey University, Palmerton, New Zealand.
Contract/grant sponsor: Swedish National Council for Engineering Sciences.
Contract/grant sponsor: SSF Program Cell Factory.
Contract/grant sponsor: NSF; contract/grant number: MCB 93-16625.
Abbreviations used: BSA, bovine serum albumin; DMSO, dimethyl sulfoxide;
g3p, minor coat protein 3; HRP, horseradish peroxidase; IPTG, isopropyl b-Dthiogalactopyranoside; moi, multiplicity of infection; OPD, orthophenylenamine; PEG, polyethylene glycol; PBS, phosphate-buffered saline; SMCC,
succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate; TB, Terrific
Broth.
Copyright # 2002 John Wiley & Sons, Ltd.
where the ability to handle large numbers of molecular
entities is one of the major advantages. Efforts to achieve
this has led to the ability to link antigen recognition to phage
replication (Dueñas and Borrebaeck, 1994; Dueñas et al.,
1996a), called selective infection (Krebber et al., 1995,
1997), which also holds great potential to rapidly identify
novel gene products. Selective infection converts the normal
phage infection of E. coli into an event dependent on a
specific protein–protein interaction. This can be achieved by
first displaying, for example, an antibody library on a phage
containing a deletion in gene 3, rendering the phage noninfective. Subsequently, a fusion protein, consisting of the
antigen/novel gene product and the part of phage g3p that
mediates infection, is allowed to interact with the antibodies. Consequently, only phage displaying an antigenspecific antibody are given the capability to infect bacteria
and thus to replicate and to be clonally amplified (Fig. 1),
thus mimicking the humoral immune response where only
antigen-specific B cells are activated.
To further understand the mechanisms of selective phage
infection we have investigated the dependence of this process on the affinity of the interacting pairs. We demonstrate
that the g3p deleted R408 helper phage has a much better
performance compared to the more common VCSM13
phage, expressing truncated g3p, when used in selective
infection and that affinity is an important discriminating
parameter.
EXPERIMENTAL
Bacterial strains
The following E. coli strains were used for the cloning,
propagation and infection experiments: Top10F', TG1,
XL1-Blue, K561 and K1762 = K561 (pJARA112,
28
N. NILSSON ET AL.
Figure 1. Schematic representation of the difference between (a)
wild-type phage, (b) phage displaying an antibody fragment, and
(c) phage engineered to perform a selective infection, the latter
strictly dependent on a p3N1N2-antigen fusion protein to
mediate the infection. N1, ®rst domain of g3p; N2, second
domain of g3p; CT, C-terminal domain of g3p; scFv, variable
domains of antibody; Ag, antigen.
pJARA131; for details on genetic markers see Nilsson et al.,
2000). The cultures were maintained in Terrific Broth (TB)
medium (10 g Bacto tryptone, 1 g yeast extract, 4 g NaCl
and 1 g glucose per liter) supplemented with appropriate
antibiotic and 1% of glucose.
The R408 g3p deleted helper DNA bacteriophage
(R408d3) was propagated in E. coli strain K1762 (Rakonjac
et al., 1997). The two vectors in K1762 complement the
deleted helper phage through the expression of g3p, which is
under the control of a phage shock protein promoter (psp).
The psp-promoter is activated in the presence of bacteriophage g4p, and is therefore silent until infection (Brissette et
al., 1991). The R408d3 helper phage prepared in a complementing host can carry out infection, since it displays
wild-type g3p on the surface. It lacks the gene coding for
protein 3, therefore it cannot produce any phage particles in
the absence of complementing C-terminal part of g3p
(Rakonjac et al., 1999). The VCSM13 mutant helper bacteriophage Dg3N was kindly supplied by K. Janda (Gao et
al., 1997). The Dg3N phage was constructed by a complete
PCR amplification of the VCSM13 genome, excluding the
N-terminal domain of g3p leaving amino acid residues 1–38
(a portion of N1) and 252–406 (C-terminal domain) of g3p
intact. The Dg3N helper phage is not infectious and needs to
be transformed into the bacterial cells in order to make
phage particles.
The phagemid vector pPAN was modified from pFAB60
(a kind gift from J. Engberg; Johansen et al., 1995) to
display scFv fragments fused only to the C-terminal domain
of g3p (amino acid residue 256–406). Cystatin C and single
chain Fv fragments against CMV epitope AD2 (AE11) and
FITC (FITCE2) were cloned into pPAN, using appropriate
cloning sites. All constructs were verified by DNA
sequencing using BigDye2 Terminator Cycle Sequencing
kit (Perkin Elmer, Elmerville, CA).
Protein-displaying, non-infectious, phagemid-containing
phage stocks were prepared following Nilsson et al. (2000).
Briefly, fresh overnight cultures of single bacterial colonies
Copyright # 2002 John Wiley & Sons, Ltd.
containing different phagemids were grown to logarithmic
growth phase, the cells were infected with R408d3 helper
phage at multiplicity of infection (moi) of 10–100. Adding
1 mM isopropyl b-D-thiogalactopyranoside (IPTG) induced
the expression. Unbound input phage was removed by
centrifugation (2000g). The pellet was resuspended in TB,
containing proper antibiotic and 1 mM of IPTG. The culture
was further propagated for 6 h at 30 °C with aeration.
When Dg3N was used to produce scFv or cystatin C
displaying non-infectious phage stocks, phagemid vectors
together with Dg3N double-stranded DNA were transformed into Top10F' bacterial cells, using electroporation.
Double-transformed bacterial cells were selected on plates
supplemented with ampicillin, kanamycin (40 mg ml 1) and
1% of glucose. Fresh over night cultures from single
colonies of these bacteria were diluted 100 times and grown
to an OD660 of 0.1. Kanamycin and IPTG (1 mM) was then
added to the cultures. At OD660 = 0.4 the concentration of
IPTG was increased to 1 mM. Expression proceeded over
night at 30 °C on a shaker.
The expression supernatants were filtered through 0.2 mm
filter. The phages were precipitated overnight at ‡4 °C with
5% polyethylenglycol (PEG) 6000 and 0.5 M NaCl. After
centrifugation at 16 000g for 30 min, the phage pellets were
resuspended in sterile phosphate-buffered saline solution
(PBS), giving a 100-fold concentration of phage.
Phage titers
ELISA determined the titer of the deleted helper phage
stocks, displaying different scFv fragments. Microtiter
plates were coated overnight at ‡4 °C with 100 ng/well of
different ligands, i.e. AD2, FITC and papain. MUC-1, an
unrelated peptide, was used as a negative control. Coated
plates were blocked with 0.1% bovine serum albumin
(BSA) in PBS. After washing, the plates were incubated
with dilutions of the PEG-precipitated phage stocks,
displaying AE11-scFv, FITCE2-scFv or cystatin C. Bound
phage were detected using horseradish peroxidase (HRP)
conjugated anti-M13 antiserum from Pharmacia (Uppsala,
Sweden), diluted 1/5000 in PBS. Orthophenylenamine
(OPD) and hydrogen peroxide were added to each well, as
chromogen and substrate. The reaction was stopped after
15 min by adding 150 ml of 1 M sulphuric acid and the
absorbance was measured at 490 nm.
Fusion proteins
The amino acid residues 1–217 of the mature g3p (the
N-terminal domains) were cloned into the heat-inducible
expression vector pHDp3 that contains a His6 tag followed
by a cysteine at the C-terminus. Expression was induced at
42 °C for 6 h and the expression supernatant was filtered and
supplemented with NaN3. The supernatant was dialyzed
against a 20 mM sodium acetate (NaAc) buffer (pH 5.5) and
applied to an anion exchange column. The g3p sample was
eluted using 100 mM sodium chloride (NaCl) and dialyzed
against a 20 mM phosphate buffer (20 mM Na-phosphate,
0.5 M NaCl, 8 mM imidazole, pH 8.0). Thereafter, the
sample was applied onto Ni nitriloacetic acid (NTA)
J. Mol. Recognit. 2002; 15: 27–32
SELECTIVE INFECTION OF E. COLI
agarose (Qiagen Ltd, Crawley, UK). More than 95% pure
g3p N-terminal (p3N1N2) was eluted, using 100 mM
imidazole, as determined by SDS–PAGE and silver
staining.
Purified p3N1N2 was used for the chemical cross-linking
of FITC and papain. For the conjugation of p3N1N2-FITC,
50 ml of a 1 mg ml 1 solution of the fluorescent probe was
added to 1 ml of the sulfhydryl containing p3N1N2 (at
2 mg ml 1) and incubated for 4 h at room temperature in the
dark. Unreacted FITC probe was removed by extensive
dialysis against PBS. The sample was loaded onto a
Sepharose column, containing covalently coupled antiFITC IgG (Sigma). Bound p3N1N2-FITC was eluted, using
0.1 M glycine-HCl (pH 2.8), and evaluated using anti-FITC
Abs (Sigma).
For the conjugation of p3N1N2-papain, papain (Sigma)
was dissolved in activation buffer (20 mM EDTA, 30 mM
mercaptoethanol, pH 2.3) and applied onto an affinity
column that had been equilibrated with separation buffer
(20 mM EDTA, 10 mM mercaptoethanol, pH 4.3). Papain
molecules were eluted using 0.1 M Na2HPO4 buffer, containing 1 mM EDTA, and 2 mM cystein was added to the
sample. The papain was then inactivated through the addition of 0.5 M iodide acetic acid to a final concentration of
0.01 M and dialyzed against PBS overnight. Inactivated and
affinity-purified papain was mixed with 10 mM succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC) in dimethyl sulfoxide (DMSO) and allowed to
react for 1 h before the removal of unbound SMCC through
dialysis against PBS. A 10 M excess of the papain-SMCC,
over the sulfhydryl containing p3N1N2, was mixed and
incubated for 3 h at room temperature. The p3N1N2-papain
sample was evaluated using SDS–PAGE and silver staining.
The AD2 fragment was amplified using standard PCR
and cloned into the pUC19 expression vector, using
appropriate restriction sites, downstream of amino acid
residue 1–217 of the mature g3p. The construct was
preceded by the g3p signal peptide and followed by six
histidine residues. All constructs were verified by DNA
sequencing using BigDye2 Terminator Cycle Sequencing
kit (Perkin Elmer). The protein was expressed and purified
from the periplasmatic space of E. coli Top10F' cells and
absorbed onto Ni-NTA agarose and eluted in 50 mM
NaH2PO4, pH 8.0, 300 mM NaCl and 250 mM imidazole.
The protein sample was analyzed in western blot, using antiAD2 scFv.
Inhibition analysis
Infection of F‡TolA‡ bacteria (strain Top10F') using wildtype phage (VCSM13) was performed in the presence of the
different fusion proteins p3N1N2, p3N1N2-AD2, p3N1N2FITC and p3N1N2-papain, respectively. Different amounts
of these proteins were used to assay their ability to inhibit
the infection process. The infections were performed using
100 ml of a log phase culture (OD660 = 0.4–0.6) of Top10F'.
The bacterial cells were preincubated with 10 ml of increasing concentrations of fusion protein at 37 °C for 15 min
and under moderate shaking (70 rpm). Thereafter, approximately 2000 VCSM13 (kan1) phage particles were added to
the bacterial cells and allowed to infect at 37 °C for 10 min.
Copyright # 2002 John Wiley & Sons, Ltd.
29
The samples were plated on TB plates containing kanamycin to detect infected cells. The plates were incubated
overnight at 37 °C.
Selective infections
Protein-displaying, non-infectious, phagemid-containing
phage particles (107–108 phage/selection) were pre-incubated for 3 h at room temperature with a series of increasing
concentrations of cognate ligand fused to the N1N2 domains
of g3p. (The final concentration of fusion protein was 0.1 pM
to 1 mM.) To these samples, 100 ml of a log phase culture of
Top10F' was added and phages were allowed to infect for
30 min at 37 °C. Unbound input phages were removed by
centrifugation for 10 min at 2000g. The bacterial cells were
resuspended in 100 ml of TB medium and plated on TB
supplemented with tetracycline, amplicillin and 1% glucose. The plates were incubated overnight at 37 °C, before
the number of colonies was enumerated.
RESULTS
The fusion proteins will constitute a physical linkage
between the non-infectious scFv/cystatin C-displaying
phage and the F pilus of the bacterial cell (Fig. 1). Consequently, the fusion proteins must functionally be capable of
mediating infection. Therefore, the ability of the fusion
proteins to efficiently block the infection of wild-type
bacteriophage was evaluated in inhibition assays. All fusion
proteins contained amino acid residues 1–217 of the mature
g3p [the N1-domain, the first glycine-rich linker (L1) and
the N2-domain]. All proteins, i.e. p3N1N2, p3N1N2-AD2,
p3N1N2-FITC and p3N1N2-papain, were capable of
effectively inhibiting the infection of wild-type phage at
micromolar concentrations (Fig. 2), indicating that the
folding of the g3p counterpart in the fusion was preserved.
Controls such as an irrelevant protein (BSA) or the specified
antigens alone could not inhibit infection (Fig. 2 and data
not shown). Furthermore, the antigenic part of the fusion
proteins was able to specifically interact with the scFv or
cystatin C displaying phage stocks in ELISA (data not
shown).
In accordance with Rakonjac et al. (1997) we observed
that the gene 3-deleted helper phage R408 (Russel et al.,
1986) generated significantly lower background infectious
transducing particles as compared to the M13KO7-derived
VCSM13 helper phage. Gao et al. (1999) recently reported
on the successful use of N1N2-deleted VCSM13 phage
(Dg3N) when selecting for catalytic antibodies. We found
that both phages gave stable phage titers (data not shown),
and R408d3 consistently gave titers that were 103–104 times
higher compared to the best titres achieved with M13KO7
(Dueñas and Borrebaeck, 1995).
There is an obvious dependence on affinity in the
interaction between the scFv and its corresponding antigen.
To evaluate this dependence we chose three different
interacting pairs having different affinities and displayed
them on the surface of two different deleted helper phage
(R408d3 and Dg3N), which also display differently (Fig. 3).
The weakest interacting pair, p3N1N2-AD2/AE11scFvJ. Mol. Recognit. 2002; 15: 27–32
30
N. NILSSON ET AL.
Figure 2. Ability of the fusion proteins to inhibit M13 phage infection. The different
fusion protein preparations were analysed for their ability to inhibit wild-type infection.
Without the presence of any fusion protein, 2000 infection events were obtained.
Different concentrations of fusion proteins were added to the indicator bacteria prior to
the phage infection, and the number of colonies was determined (representative data
from one experiment out of total 12).
phage (KA = 6 10 7 M, Lantto and Ohlin, unpublished
observation), was analyzed for selective infection, using
10 6–10 9 M concentration of the fusion protein [Fig.
4(A)]. An approximately 10-fold increase in the number
of colony forming units (cfu) was obtained, at a concentration of 10 8 M of the p3N1N2-AD2 fusion protein in
combination with the AE11scFv-displaying Dg3N phage or
at a concentration of 10 9 M together with the AE11scFvdisplaying R408d3 phage. Here the inhibitory effect of the
fusion protein can clearly be seen at concentrations above
10 8 M (see also Fig. 2), giving a decreased efficiency in
selective infection [Fig. 4(A)]. No selective infection could
at any time be achieved with the Dg3N phage at
concentrations below 10 8 M of fusion protein. The FITC/
FITCE2scFv-phage (KA = 3 10 10 M; Vaughan et al.,
1996) was analyzed for selective infection, using 10 10–
10 13 M concentration of the fusion protein [Fig. 4(B)]. At a
concentration of 10 10 M of the fusion protein p3N1N2FITC, in combination with R408d3-generated FITCE2scFvdisplaying phage, a 2.5 102 fold increase in the relative
Figure 3. Schematic representation of the two different helper phage systems. The deleted gene sequences of
respective helper phage are indicated in the ®gure (star). The R408d3 phage displays a wild-type g3p if produced by
the bacterial cell, while the Dg3N helper phage displays the C-terminal domain of g3p (amino acid residue 252±406)
together with the amino acid residues 1±38 (®rst part of N1). Furthermore, as an antibody-displaying phage, the
R408d3 displays only antibody and the Dg3N phage a combination of the antibody and the C-terminal domain of g3p.
Copyright # 2002 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2002; 15: 27–32
SELECTIVE INFECTION OF E. COLI
31
number of cfu was observed. A selective infection was
detected even at the lowest concentration of 10 13 M fusion
protein. Using this interacting pair, no selective infection
could at any time be achieved with the Dg3N phage. Finally,
the interacting pair with the highest affinity, p3N1N2papain/cystatin C-phage (KA of around 1 10 11 M; Pol et
al., 1995), gave the highest increase of cfu at the lowest
concentration of fusion protein during the selective infection [Fig. 4(C)]. A 2 103 fold increase in the relative
number of cfu was detected when using 10 10 M of the
p3N1N2-papain fusion protein together with R408d3generated cystatin C particles. Again, using Dg3N phage,
no selective infection could at any time be achieved with
this interacting pair.
DISCUSSION
Figure 4. Selective infection depends on the af®nity of interacting
proteins. The two deleted helper phages (R408d3 and Dg3N) were
utilized to generate antibody-displaying phage particles, used for
selective infection. Three interacting pairs were used to demonstrate the dependence on fusion protein and the af®nity between
the pairs: (A) scFv-fragment AE11 (anti-CMV antibody) binds to
p3N1N2-AD2; (B) scFv-fragment FITCE2 (anti-FITC antibody)
binds to p3N1N2-FITC; (C) cystatin C fragment binds to
p3N1N2-papain. The data is presented as the ratio of number of
colonies obtained with different concentrations of fusion protein
to the number of colonies obtained in the absence of fusion
protein (representative data from triplicate runs). The darker bars
are data generated with the Dg3N helper phage, while the lighter
bars are data generated using the R408d3 helper phage.
Copyright # 2002 John Wiley & Sons, Ltd.
Selective infection is the first example of how a biological
interaction, the infection of E. coli by a filamentous phage,
has been utilized to study protein–ligand interactions not
involved in the infective event. The principle is to generate a
library of protein variants (often antibody fragments) fused
to the C-terminal domain of g3p (p3C), thus creating noninfectious phage particles (Dueñas and Borrebaeck, 1994;
Krebber et al., 1995). Binding to a fusion protein then
makes specifically interacting members of this phage library
infectious. The fusion protein consists of the antigen and the
N1N2 domains of g3p [Fig. 1(c)]. This approach has been
used to study optimal folding properties (Pedrazzi et al.,
1997), kinetic parameters (Dueñas et al., 1996a) and the
humoral immune response in vitro (Dueñas et al., 1996b). It
has also been shown to achieve specific enrichment factors
of 1010 after only two rounds of selection (Dueñas and
Borrebaeck, 1994). The principle of using specific proteinligand interactions that result in a biologically measurable
event (Malmborg et al., 1997) has great potential in, for
example, functional genomics, where there is a need to
rapidly characterize novel gene products or for in vitro
evolution where very high binding affinities are desired.
However, to use selective infection as a tool in these
applications we need to understand what effect the affinity
of the interacting pairs has on the process.
The affinity of the interacting pairs is an important
functional factor, which has not been studied before. To
investigate the role of affinity we selected three different
binding pairs exhibiting affinities in the range of 107–
1010 M 1 and used two different gene 3-deleted helper
phages to generate the non-infectious antibody displaying
phages. Using R408d3, we can show a direct correlation
between binding affinity and the efficiency of the selective
infection (Fig. 4). There is also a correlation between the
concentration of fusion protein and the number of selective
events, shown in the titrations. Due to the demonstrated
inhibitory effect of the fusion protein (Fig. 2), it is important
to use several concentrations of fusion protein during selection. The efficiency of selective infection is also demonstrated to be higher when using higher affinity interacting
pairs (Fig. 4). Therefore, selective infection would be
particularly well suited for selection of high affinity
(108 M 1) interactions that in many cases are difficult to
find using conventional solid phase based phage selection
J. Mol. Recognit. 2002; 15: 27–32
32
N. NILSSON ET AL.
methods (Hawkins et al., 1992). Only R408d3 phage
behaved predictably during selection. When using the
Dg3N version of VCSM13, we obtained selective infection
solely with anti-CMV scFv. The slight shift in the peak
efficiency seen in Fig. 4(A) might result from the fact that
the display is more efficient with R408d3 than with Dg3N.
The Dg3N helper phage itself codes for a truncated version
of g3p that will be competing for incorporation in the phage
coat with the scFv molecules (Fig. 3). Thus, in the case of
R408d3, all the released phage particles display several
copies of scFv on the surface, while in the case of Dg3N the
display could range from zero to five copies of the desired
antibody fusion, depending on stability of the scFv-p3C
fusion in the cells, its toxicity and differential stability of the
phagemid. Despite the higher affinity between FITCFITCE2scFv and papain/cystatin C, no specific infection
could be achieved at any time using Dg3N. The reason for
this is still not clear. However, if it is necessary to have more
than one copy of g3p on the surface of phage in order to get
the phage DNA translocated into the bacterial cytoplasm
(Rakonjac et al., 1999), the actual number of displayed
protein-p3C fusions would be a crucial parameter for
successful selective infection.
In summary, selective infection is dependent on the
affinity and avidity in the interaction between the proteindisplaying phage particle and the cognate ligand. This was
demonstrated for the first time, also showing that polyvalent
display using R408d3 phage performed adequately in all
respects.
Acknowledgements
This investigation was supported by a grant from the Swedish National
Council for Engineering Sciences, the SSF Program Cell Factory and by
NSF grant MCB 93-16625.
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