FEMS Microbiology Letters 130 (1995) 293-300
Overproduced and purified receptor binding protein pb5 of
bacteriophage T5 binds to the T5 receptor protein FhuA
M. Mondigler, R.T. Viigele, K.J. Heller
Universitiit Konstanz,
*
Fakultiitfir Biologic, Postfach 5560 (M605), D-77484 Konstanz, Germany
Received 17 May 1995; revised 29 May 1995; accepted 31 May 1995
Abstract
A promotor-less
oad gene of bacteriophage T5, encoding the receptor binding protein pb5, was cloned into pT7-3 under
the control of phage T7 promoter @lo. Induction with IF’TG resulted in enhanced production of pb5. Upon fractionation of
the producing cells, most of the overproduced pb5 was found in the membrane fraction, which was most likely due to
aggregation of the protein. The minor, soluble fraction of pb5 specifically inhibited adsorption of TS to its FhuA receptor
protein. Inhibition was also seen with trace amounts of pb5, and binding of pb5 to Fhu4 appeared to be almost irreversible.
Purification of pb5 from the cytosolic fraction was performed by FPLC using a MonoQ column. pb5, which did not bind to
the matrix of the column, was obtained in almost pure form. The purified protein also inhibited T5 adsorption.
Keywords:
Bacteriophage T5; Escherichia coli; oad Gene; pb5 Receptor binding protein
1. Introduction
Bacteriophages recognize
specific interaction of phage
exposed bacterial structures.
ses of interactions between
host bacteria by highly
proteins with sutfaceSo far molecular analy-
receptors and receptor
binding proteins have mostly relied on in vivo studies like isolating and characterizing phage with altered adsorption characteristics [ 11. For filamentous
phage this has been done by directly manipulating on
the phage genome the gene encoding the receptor
l
Corresponding author. Present address: Institut fiir Mikrobi-
ologie, Bundesanstalt fiir Milchforschung,
Postfach 6069, D-24121
Kiel, Germany. Tel.: +49 (431) 609 340; Fax: +49 (431) 609
222.
037%1097/95/$09.50
0 1995 Federation
.WDI 0378-1097(95)00221-9
of European
Microbiological
binding protein [2,3]. The strategy has been possible
due to the small size of the genome which can be
handled like a plasmid. For complex, tailed phages a
different strategy has been applied. It relied on the
isolation of phage-resistent bacterial mutants still
expressing the receptor. Using these mutants, hostrange mutants of the phage were isolated and the
alterations within the phage genomes were characterized [4-61.
For the complex, tailed phage T5 we wanted to
use a different strategy. Since we had cloned the
gene encoding the receptor binding protein pb5 [7],
we wanted to manipulate the gene and study the
interaction of the altered gene product with the receptor, the FhuA Protein [8]. However, an essential
prerequisite for such experiments is that pb5 expressed from a plasmid can bind to FhuA in its free
form.
Societies.
All rights reserved
294
M. Mondigler et al. / FEMS Microbiology Letters 130 (1995) 293-300
In this communication
we show that overproduced, purified pb5 can bind to FhuA. We further
show that interaction between pb5 and FhuA can be
studied without prior purification of pb5 and that
binding of pb5 to FhuA appears to be comparably
stable, as the binding of phage T5 to FhuA.
resuspended in 200 ~1 of phosphate-buffered
saline
(PBS). The supematant was centrifuged at 5000 X g
for 20 min to sediment membranes. The membrane
pellet was resuspended in 100 ~1 of PBS, the supernatant contained the soluble cytosolic fraction.
2.4. Purification
2. Materials and methods
2.1. Bacteria, phages, plasmids,
media and growth
conditions
E. coli BL21tDE3)pLysE
[9] (named BL21
throughout this study) was used for overexpression
of proteins under the control of the phage T7 @lo
promoter. BL21/5
is a T5 resistant derivative of
BL21. E. cofi AI32847 [lo] was used for the phage
adsorption assays described below.
Phages T5 and BF23 have been described before
[ll], as have been media and growth conditions [12].
Plasmids used were pT7-3 [14] and pVK88 [7].
pVK88 contains a promoter-less oad gene of phage
T5 cloned under the control of the phage T7 @lo
promoter of pT7-3.
2.2. Electrophoresis
of proteins
Proteins were separated by SDS-PAGE according
to the method described by Laemmli [13]. Gels with
acrylamide concentrations between 8 and 12% were
used. After separation, proteins were stained with
Coomassie brilliant blue.
2.3. Overexpression
of proteins
For expression of oad cloned into the pT7-3
promoter plasmid [14], E. coli BL21 or its T5
resistant mutant BL21/5 were transformed with the
respective plasmids and grown overnight in LB
medium supplemented with 50 pg Ap per ml. The
culture was diluted 1:lOO in the same medium. At an
A 578 of 0.3, expression of oad was induced by
addition of IPTG at a final concentration of 2 mM.
Cells were harvested after 2 h of induction, concentrated lOO-fold, disrupted in a french press, and
fractionated by centrifugation.
Cell debris was sedimented by centrifugation at 1000 X g for 10 min and
of pb.5
E. coli BL21, transformed
with pVK88, was
grown in 200 ml M9 medium supplemented
with
0.4% casamino acids, 1 mM MgSO,, 0.1% glucose,
and 100 pg/ml
ampicillin. At an AST8 of 0.3 expression of oad was induced by addition of 2 mM
IPTG. Three hours later the cells were harvested by
centrifugation for 10 min at 10000 X g and 4°C. The
pelleted cells were resuspended in 5 ml of 50 mM
Tris/HCl,
pH 8.0. After four cycles of french pressure treatment (10000 psi), insoluble material was
removed by centrifugation for 1 h at 40000 X g and
4°C. The supematant
material was applied to an
FPLC MonoQ-column
equilibrated
with 50 mM
Tris/HCl,
pH 8.0. Proteins were eluted from the
column first with 50 mM Tris/HCl,
pH 8.0, and
then with a gradient of O-O.5 M NaCl within the
same buffer.
2.5. Phage adsorption
assay
An overnight culture of E. coli AB 2847 grown
in LB medium was diluted 1:lOO in prewarmed LB
medium. At an A578 of 0.3, cells from 2 ml of this
culture were sedimented by centrifugation and resuspended in 100 ~1 of pb5-containing
soluble fraction.
After preincubation at 30°C for 15 min, 10 ~1 of a
T5 lysate (titer: 3 X lo7 per ml) were added to 90 ~1
of preincubated cells. Two, 5, 10 and 20 min after
addition of phages 10 ~1 aliquots were taken, diluted
500-fold in icecold PBS, vortexed intensively to stop
phage adsorption, and centrifuged at 1000 X g for 5
min to sediment cells together with adsorbed phage.
100 ~1 aliquots of the supematant fluid, containing
free unadsorbed phage, were 200 ~1 of E. coli
AI32847 (A,,, = 1.0) and poured onto TB agar
plates.
2.6. Phage inactivation
assay
10 ~1 of a phage lysate (titer: 3 X lo7 per ml)
were incubated at 30°C with 90 ~1 of different
M. Mondigler et al. / FEMS Microbiology Letters 130 (1995) 293-300
dilutions of the soluble fractions containing pb5.
After 2, 5, and 10 min, 10 ~1 aliquots were taken,
diluted 500-fold in icecold PBS, and vortexed intensively. 100 ~1 aliquots of these dilutions were mixed
with 5 ml TB top agar containing 200 ~1 of E. coli
Al32847 (A,,, = 1.0) and poured onto TB top agar
plates.
295
structures by self-assembly. On the other hand, overproduced proteins often form intracellular aggregates, termed ‘inclusion bodies’, which contain the
protein in a denatured, biologically inactive form
[16]. Since we were interested in a biologically
active form of overproduced pb5, we focused on the
cytoplasmic, soluble form of pb5 for further experiments.
2.7. Stability of binding of pb5 to the receptor
3.2. Binding of overproduced pb5 to its FhuA recepThis assay was performed analogous to the phage
adsorption assay. After 15 min of incubation with
pb5, the cells were sedimented, the entire supernatant was carefully removed, and the cells were
resuspended in the same volume of PBS and incubated for 5 min. Centrifugation and incubation in
PBS was repeated twice. After each incubation, 90
~1 aliquots were used for phage adsorbtion assays,
as described above.
3. Results and discussion
3.1. Overexpression
of the phage T5 receptor bind-
ing protein pb5
The oad gene of phage T5 expressing the receptor binding protein pb5 has recently been cloned [7].
For overproduction of pb5 with the aid of the T7polymerase/promoter
system [14], the oad gene,
carried on a 2693 bp HindIII-PvuII fragment of
pVK3BIO [7], was subcloned into pT7-3 to yield
pVK88 (Fig. 11, which was transformed into E. coli
BL21. After induction with IPTG, expression of pb5
was monitored by SDS-PAGE of total cellular protein at different times after induction. Based on these
results induction with 2 mM IPTG for 2 h was used
for further experiments (data not shown).
To analyze the cellular distribution of pb5, induced cells were harvested, desintegrated in a french
pressure cell, and separated into a debris, cytoplasm
and membrane fraction by centrifugation. Only a
small amount of the overproduced pb5 was found in
the cytoplasmic fraction, the largest amount was
present in the membrane fraction (Fig. 2). This result
is not surprising. On the one hand, structural phage
proteins tend to aggregate [15], probably as a consequence of their natural property to build up phage-
tor protein
To test whether overproduced pb5 was capable of
binding to FhuA, we wanted to develop a simple
assay which was based on the blocking of FhuA for
further interaction with T5 by pb5 present in the
crude cytoplasmic preparation. However, to exclude
unspecific blocking by other components, several
control experiments had to be carried out. In all these
experiments phage BF23 was used as a control.
BF23 is closely related to T5 but uses the BtuB outer
membrane protein as a receptor [11,17] instead of
FhuA.
First, we tested whether T5 was affected by substances present in the soluble fraction of either
BL21[pT7-31 or BL21[pVK88]. Incubation for 10
min at 30°C with undiluted soluble fraction of
BL21[pT7-31 resulted in almost 100% inactivation of
T5. No inactivation was seen when the soluble fraction of BL21[pVK88] was applied. In addition, no
inactivation by the soluble fraction of BL21[pT7-31
was seen for BF23 Table 1. Since BL21 cells lack
the BF23 receptor protein in the outer membrane
(Mondigler and Heller, unpublished results), the data
indicated that soluble, unsedimented FhuA receptor
particles were responsible for inactivation of T5 by
the soluble fraction of BL21[pT7-31. This was verified by using the fhuA strain BL21/5 for isolation
of soluble fractions: no inactivation of T5 occurred
in this case (Table 1).
The second control experiment was designed to
rule out unspecific binding to the outer membrane of
E. coli of substances present in the soluble fractions
of BL21[pT7-31 or BL21[pVK88], which could make
the receptors inaccessible for phage. We used BF23
as a tester phage. No inhibition of BF23 adsorption
was seen when the cells used for adsorption assays
were preincubated with the soluble fractions of
296
M. Mondigler et al. / FEMS Microbiology Letters I30 (1995) 293-300
IS1 p
2.7 kb HindIll-hull
j
---
fragment
of pVK3/lSl-1
2.4 kb Hindlll-Smal
fragment
of pT7-3
!
H
pVK88
pT7-3
(P/S)
+_
.~~~~
_ ._~ IS1
4
@I 0 promoter
H
’
bla
Fig. 1. Physical and genetic map of pVK88. pVK88 was constructed by cloning into pT7-3 [14] a 2.7 kb PuuII-Hind111 fragment of
pVK3/ISl-1
[12]. The fragment, obtained by cleaving at the PouII site within the IS1 and the Hind111 site 71 bp downstream of oad,
contains a promoterless oud gene and 678 bp of the IS1 element. Thus, the oad gene in pVK88 is exclusively under the control of the T7
@lo promoter. DNA fragments of different origins (T5, ISl, and pT7-3) are indicated by differently shaded boxes. Directions of
transcription are indicated by arrows for oad and bla. Restriction enzyme cleavage sites are: A: ApaI; B: BumHI; Bc: EC/I; Bg: BglII; H:
HindIII; P: PuuII; S:SmaI; SC: SacI; X: XhoI. Sites in brackets are no longer accessible to the respective enzymes.
cated that pb5 within the crude cell lysate was most
likely capable of blocking the soluble FhuA receptor
particles. This was proven by the result that E. coli
AB2847, preincubated for 15 min with the soluble
BL21[pT7-31 or BL21[pVK88], respectively. Thus,
unspecific inhibition of adsorption was ruled out.
The fact that no inactivation of T5 was seen by
the soluble fraction of BL21[pVK88], already indi-
kDa
Ma
b
pT7-4
c La
_____
b
c
pVK88
Fig. 2. Cellular localization of overproduced pb5. Exponentially growing cultures of BL21/5[pT7-31
and BL21/5[pVK88]
were induced for
2 h with 2 mM IPTG. Cells were harvested and separated into a debris (a), membrane (b), and cytoplasmic fraction (c) as described in
Materials and methods. The fractions were subjected to SDS-PAGE. On the left the positions and molecular masses of marker proteins are
indicated. The position of pb5 is shown in the right margin. (MI marker proteins.
M. Mondigler et al. / FEMS Microbiology Letters 130 (1995) 293-300
Table 1
Effect of soluble fractions
of BL21 cells on viability
of phages T5 and BF23
Number of phages (%) after incubation
fraction of different BL21 strains a
Relative amount
of soluble fraction
BL21[pT7-41
1:l
1:lO
1:lOO
0 (PBS)
with soluble
BL21[pVK88]
BF23
T5
2
32
110
100
T5
103
115
90
100
BF23
127
110
99
100
a Phage numbers higher than 100% are most likely due to dissolving
fraction of BL21/5[pVK88], was blocked for adsorption of phage T5: the titer of free phages in the
adsorption assay stayed constant during the whole
period of incubation (Fig. 3). In contrast, the soluble
fraction of BL21/5[pT7-31 did not show any effect
on T5 adsorption: after 30 min almost all of the
phage added were adsorbed to their host cells (Fig.
3). Together with the fact that BF23 was not inhibited by the soluble fractions of either BL21/5[pT7-31
or BL21/5[pVK88], these experiments demonstrated
that the soluble fraction of BL21/5[pVK88] con-
unadrorbed Dhape 1%)
l~,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,i
02
5
10
15
20
25
297
30
2s
time (mid
Fig. 3. Inhibition of T5 adsorption by overproduced cytosolic pb5.
T5 adsorption was analyzed to E. coli AB2847 cells preincubated
with either cytosolic fraction from BL21/5[pT7-31
(Cl) or BL21/5
[pv~88] ( + ), as described in Materials and methods.
BL21/5[pT7-41
BL21/5[pVk88]
T5
T5
130
125
122
100
of phage aggregates
93
91
82
100
109
98
104
100
by soluble fraction.
tained a component which was able to bind to the
FhuA receptor protein. Since this component was not
present in the soluble fraction of BL21/5[pT7-31, it
was most likely pb5.
3.3. Purification of pb5 overproduced from BL21/
5w7w
To verify that pb5 was indeed responsible for the
FhuA-blocking activity present in the soluble fraction of BL21/5[pVK88], we tried to purify pb5 and
demonstrate blocking activity for the purified protein.
200 ml of an E. coli BL21[pVK88] culture were
induced for synthesis of pb5 by addition of 2 mM
IPTG for 2 h. After concentration and disruption of
the cells, followed by removal of insoluble material
(including aggregated pb5), proteins of the soluble
fraction were separated on a MonoQ column by
FPLC. A protein corresponding in size to pb5 did not
bind to the MonoQ matrix and was eluted within
fractions nos. 2 and 3 in almost pure form. Only one
other protein of approx. 20 kDa was detectable in
these fractions (Fig. 4).
We used several FPLC fractions to test whether
binding to FhuA of a component within the fractions
could be demonstrated. Binding to FhuA was indicated again by blocking FhuA on the cell surface for
adsorprtion of phage T5. When fractions 2 and 3
from the FPLC separation were used, they exhibited
blocking activity (Table 2). The activity followed the
concentration of the presumed pb5 within the FPLC
fractions. Fraction 2 contained the majority of presumed pb5 and blocked T5 adsorption completely,
while fraction 3 contained about one third of the
M. Mondigier ei at. / FEMS Microbiology Letters 130 (199s) 293-300
298
Table 2
Binding of purified pb5 to FhuA
by undiluted
crude cytosol
98
Inhibition of T5 adsorption (%) a
by undiluted FPLC fraction no.
2
3
4
19
22
96
24
9
a The mean of three independent
0
23
0
experiments
0
is shown.
amount of fraction 2 and blocked T5 adsorption by
about 25%. No protein was visible in fraction 4;
however, approx. 10% of blocking activity was seen
with this fraction. The 20 kDa protein was present in
fractions 2 and 3 in about equal amounts. Other
fractions were inactive in this assay: T5 adsorption
was not reduced, it was comparable to the adsorption
to untreated control cells. These results very strongly
indicated that pb5 indeed was responsible for the
blocking activity for T5 adsorption present in the
soluble fraction of BL21/5[pVK88].
Thus at least a
fraction of the overproduced, soluble pb5 must be in
a biologically active conformation,
able to bind to
the FhuA receptor protein.
The fact that an overproduced receptor binding
protein of a phage can bind to its receptor is by no
means self-evident. In the phage tail, receptor binding proteins serve two functions: (i) they allow for
highly specific receptor-binding,
and (ii) they have to
signal successful binding to the phage machinery
needed for infection. This signalling
most likely
starts by a conformational
change of the receptor
binding protein, induced by the binding to the receptor, and is transmitted to neighbouring
proteins by
induction of conformational
changes in these proteins. Finally, these conformational changes result in
a tail structure competent for DNA passage, and in
triggering DNA release from the head (see [l] for a
review). Taking into account the intimate contact and
mutual interdependence
with respect to conformational changes of tail proteins, one could have imagined that the receptor binding protein during assembly is forced into a special conformation within the
phage tail, thereby adopting a conformation unstable
in the free, soluble form. However, our results suggest that pb5 is rather synthesized in the same conformation as it is assembled into the T5 tail.
kDa
...O’
t-
66
c-
46
c-
29
‘~‘,”
:
/;
&
I
SF
1
I
2
I
I
I
I
6
I
I
I
I
I
10
I
1
I
1
SF
1
I
16
I
I
I
I
20
I
I
I
I
I
I
24
FPLC fractions
Fig. 4. Separation by FPLC of soluble proteins of BLZl[pVK88]. Soluble proteins were obtained from BL21[pVK88] cells overproducing
pb5 and separated by FPLC on a MonoQ column as described in Materials and methods. Fractions were collected and aliquots of these were
subjected to SDS-PAGE. The numbers of the fractions are indicated in the lower margin. SF shows the soluble fraction used for FPLC
separation. The positions of pb5 (left margin) and of marker proteins (right margin) are shown.
M. Mondigler et al. / FEMS Microbiology Letters 130 (1995) 293-300
3.4. Afinity and stability of binding of pb5 to FhuA
To estimate the quantity of overproduced pb5
present in the soluble fraction, phage adsorption
assays were carried out with progressive dilutions of
the soluble fraction of BL21/5[pVK88]. A 60-fold
dilution inhibited phage adsorption with the same
efficiency as the undiluted soluble fraction. A lOOOfold dilution still caused an inhibition of phage adsorption of 20% as compared to the undiluted soluble fraction (Fig. 5).
Binding of phage T5 to its receptor has been
described to be irreversible due to covalent bond
formation [18]. However, since these bonds are not
formed between pb5 and FhuA but rather between
three copies of tail protein pb4 [19], binding of free
pb5 to FhuA may be reversible. To test the stability
of binding of pb5 to FhuA, cells preincubated with
the soluble fractions were washed with PBS up to
three times and incubated after each wash to allow
dissociation of bound pb5 from its receptor. Thereafter, T5 phage were added and adsorption was
monitored. While the number of free phage in the
supematant decreased by 90% when control cells
(preincubation with soluble fraction of BL21/5[pT731) were used, no reduction in the number of approx.
3.2 X lo7 plaque forming units per ml in the supernatant was observed when preincubated, washed cells
were used. This indicated that no dissociation of pb5
1000
no. of unadlorbed
phwe
0
0
500
”
--“\
1
10
\
\
100
I
1000
dilution factor
Fig. 5. Inhibition of TS-adsorption by different concentrations
of
pb5. E. coli AB2847 cells were incubated with different dilutions
of cytosolic fraction from BL21/5[pVK88]
and subsequently
analyzed for T5 adsorption, as described in Materials and methOdS.
299
from FhuA occured during the washing procedure.
Thus, binding of pb5 to FhuA appears to be very
stable even in the absense of covalent bond formation of pb4. This result supports data of studies on
the interaction between intact phage and purified
FhuA receptor protein [20]. These studies indicated
that the affinity of T5 for FhuA was rather high,
since nano-molar concentrations of FhuA readily
inactivated T5 phage. In the present study we did not
examine the affinity of pb5 to FhuA in particular.
However, the fact that very small amounts of pb5
were needed for receptor blocking supports the view
that the high affinity for FhuA of phage T5 is
determined by the high affinity of pb5 for FhuA.
Acknowledgements
We thank V. Krauel for construction of pVK88
and T. Holz for carrying out some of the experiments
on inhibition of T5 adsorption. This work was supported by the Deutsche Forschungsgemeinschaft (He
1375/3-3X
References
[l] Heller, K.J. (1992) Molecular interaction between bacteriophage and the gram-negative cell envelope. Arch. Microbial.
158, 235-248.
[2] Stengele, I., Bross, P., Garces, X., Gray, J. and Rasched, I.
(1990) Dissection of functional domains in phage fd adsorption protein: discrimination between attachment and penetration sites. J. Mol. Biol. 212, 143-149.
[3] Endemann, H., Bross, P. and Rasched, I. (1992) The adsorption protein of phage IKe. Localization by deletion mutagenesis of domains involved in infectivity. Mol. Microbial. 6,
471-478.
[4] Drexler, K., Dannull, J., Hindenach, I., Mutschler, B. and
Henning, U. (1991) Single mutations in a gene for a tail fiber
component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor. J. Mol.
Biol. 219, 655-663.
[S] Henning, U. and Hashemolhosseini,
S. (1994) Receptor
recognition by T-even-type coliphages. In: Molecular Biology of Bacteriophage
T4 &ram,
J.D., Ed.), pp. 291-298,
ASM Press, Washington.
[6] Riede, I., Drexler, K., Eschbach, M.-L. and Henning, U.
(1987) DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages
T2, K3 and of K3 hostrange mutants. J. Mol. Biol. 194, 31-39.
300
M. Mondigler et al. / FEMS Microbiology Letters 130 (1995) 293-300
[7] Krauel, V. and Heller, K.J. (1991) Cloning, sequencing, and
recombinational
analysis with bacteriophage
BF23 of the
bacteriophage
T5 oud gene encoding the receptor-binding
protein. J. Bacterial. 173, 1287-1297.
[8] Kadner, R.J., Heller, K., Coulton, J.W. and Braun, V. (19801
Genetic control of hydroxamate-mediated
iron uptake in
Escherichia coli. J. Bacterial. 143, 256-264.
[9] Studier, F.W. and Moffat, B.A. (1986) Use of bacteriophage
T7 RNA polymerase to direct selective high-level expression
of cloned genes. J. Mol. Biol. 189, 113-130.
[lo] Hantke, K. and Braun, V. (1978) Functional interaction of
the to4 / roti receptor system of Escherichia coli. J. Bacteriol. 135, 190-197.
[ll] Heller, K.J. (19841 Identification of the phage gene for host
receptor specificity by analyzing hybrid phages of T5 and
BF23. Virology 139, 11-21.
[12] Decker, K., Krauel, V., Meesmann, A. and Heller, K.J.
(1994) Lytic conversion of Escherichia coli by bacteriophage TS: blocking of the FhuA receptor protein by a
lipoprotein expressed early during infection. Mol. Microbial.
12,321-332.
[13] Laemmli, U.K. (19701 Cleavage of structural proteins during
the assembly of the head protein of bacteriophage T4. Nature
(Land.) 227, 680-685.
[14] Tabor, S. and Richardson, C.C. (1985) A bacteriophage T7
RNA polymerase/promoter
system for controlled exclusive
[15]
[16]
[17]
[18]
1191
[20]
expression of spcci, SCgenes. Proc. Natl. Acad. Sci. USA 82,
1074-1078.
Eamshaw, W.C., Goldberg, E.B. and Crowther, R.A. (19791
The distal half of the tail flbre of bacteriophage T4. Rigidly
linked domains and cross-p structure. J. Mol. Biol. 132,
101-131.
Marston, F.A.O. and Hartley, D.L. (1990) Solubilization of
protein aggregates. In: Guide to Protein Purification (Deutscher, M.P., Ed.), Methods in Enzymology, Vol. 182, pp.
264-276, Academic Press, San Diego.
McCorquodale,
D.J. and Warner, H. (19881 Bacteriophage
T5 and related phages. In: The Bacteriophages,
Vol. 1
(Calendar, R., Ed.), pp. 439-475, Plenum Press, New York.
Zarnitz, M.L. and Weidel, W. (1963) iiber die Rezeptorsubstanz tiir den Phagen TS. VI. Mitteilung: Die Thermodynamik der Kontaktbildung
zwischen Phage und Rezeptor
sowie deren miigliche Bedeutung
als morphogenetischer
Modellmechanismus.
Z. Naturforsch. 18b, 276-280.
Feucht, A., Heinzelmann, G. and Heller, K.J. (1989) Irreversible binding of bacteriophage
T5 to its FhuA receptor
protein is associated with covalent crosslinking
of three
copies of tail protein pb4. FEBS Lett. 255, 435-440.
Hoffmann. H., Fischer, E., Kraut, H. and Braun, V. (1986)
Preparation of the FhuA (TonA) receptor protein from cell
envelopes of an overproducing
strain of Escherichia coli
K12. J. Bacterial. 166, 404-411.