Escherichia coli ykfE ORFan Gene
Encodes a Potent Inhibitor of C-type Lysozyme*
Vincent
Monchois
§,
Chantal
Abergel
,
James
Sturgis¶,
Sandra
Jeudy
, and
Jean-Michel
Claverie
From the
Information Génétique et
Structurale, UMR1889 CNRS-AVENTIS and ¶ Laboratoire
d'Ingénierie des Systèmes Macromoléculaires, UPR
9027, 31 Chemin Joseph Aiguier, 13402 Marseille, CEDEX 20, France
Received for publication, November 13, 2000, and in revised form, February 22, 2001
 |
ABSTRACT |
The complete nucleotide sequences of over
37 microbial and three eukaryote genomes are already publicly
available, and more sequencing is in progress. Despite this
accumulation of data, newly sequenced microbial genomes continue to
reveal up to 50% of functionally uncharacterized "anonymous"
genes. A majority of these anonymous proteins have homologues in
other organisms, whereas the rest exhibit no clear similarity to any
other sequence in the data bases. This set of unique, apparently
species-specific, sequences are referred to as ORFans. The biochemical
and structural analysis of ORFan gene products is of both evolutionary
and functional interest. Here we report the cloning and expression of
Escherichia coli ORFan ykfE gene and the
functional characterization of the encoded protein. Under physiological
conditions, the protein is a homodimer with a strong affinity for
C-type lysozyme, as revealed by co-purification and co-crystallization.
Activity measurements and fluorescence studies demonstrated that the
YkfE gene product is a potent C-type lysozyme inhibitor
(Ki
1 nM). To denote this
newly assigned function, ykfE has now been registered under
the new gene name Ivy (inhibitor of
vertebrate lysozyme) at the E. coli
genetic stock center.
 |
INTRODUCTION |
Despite the accumulation of sequence information from a large
diversity of species and phyla, newly sequenced bacterial genomes continue to reveal a high proportion of genes of unknown function (1),
including a significant subset of "ORFans" (2),
i.e. putative open reading frames
(ORFs)1 without significant
similarity to any previously encountered protein (or conceptual
translation) sequences. Most genes found in data bases have only been
predicted by computer methods and never experimentally validated. It is
thus expected that some annotated ORFs, in particular among the ORFans,
might not correspond to real genes. In a previous study, we verified
the existence of a cognate transcript for 25 Escherichia
coli ORFans with a surprising rate of success (92%) (3). Given
that most ORFans appear to be transcribed, we have now initiated a
systematic expression and structure determination program for the
proteins encoded by these (apparently) unique genes. Because
three-dimensional structures are more resilient to evolution and
change than amino acid sequences, it is expected that some ORFans
should exhibit structural similarity to previously described protein
families, hence providing some functional hints. Alternatively,
targeting ORFans for structure determination is also a suitable
strategy to optimize the discovery of original protein folds, one of
the goals of structural genomics.
In a pilot study involving five ORFan genes, we succeeded in producing
four of them in E. coli as soluble proteins, and we report
here the most advanced project, ykfE. YkfE
(Swiss-Prot accession number P45552; b0220 in the Blattner data
base (4)) is a 474-nucleotide-long uncharacterized ORF. It is part of a
single gene operon and was found to exhibit a high level of expression
during the exponential and stationary phases of E. coli
growth (3). The ykfE ORF exhibits an N-terminal signal peptide cleaved to produce the mature protein (5, 6). Initial purification steps and biochemical analyses suggested a strong interaction between this protein and hen egg white lysozyme (HEWL). The
existence of a stable complex was confirmed by biophysical analyses,
and enzymatic studies revealed the capacity of ykfE to inhibit hen and
human C-type lysozymes through a specific interaction. The x-ray
structure determination of ykfE, both in isolation (7) and in a complex
with HEWL, is currently in progress and should allow us to understand
the molecular basis of the ykfE-lysozyme interaction at atomic
resolution. To denote its newly assigned function, ykfE has
been registered under the new gene name Ivy (for
inhibitor of vertebrate lysozyme)
at the E. coli genetic stock center.
 |
EXPERIMENTAL PROCEDURES |
Cloning of ykfE--
The 474-base pair ykfE ORF
including its own signal peptide was polymerase chain reaction
amplified from E. coli K-12 MG1655 genomic DNA using pwo DNA
polymerase (Roche Molecular Biochemicals). Primer
sequences, 5'-TTATACCATGGGCAGGATAAGCTC-3'
(sense) and
5'-GCTAAAGATCTAAAATTAAAGCCATCCGGA-3'
(antisense) with NcoI (sense) and BglII
(antisense) sites (underlined), were used. After digestion
with NcoI + BglII, the polymerase chain reaction product was cloned into a pQE-60 vector (Qiagen) to express
ykfE in phase with a C-terminal His6 tag
(plasmid pQE-0220).
Expression and Purification of the YkfE Gene Product--
The
ykfE gene product (Ivy) was expressed by culturing E. coli
XL1-Blue carrying the plasmid pQE-0220 in LB + Amp medium. After
initial growth at 37 °C, temperature was set at 30 °C when A600 reached 0.4, and Ivy expression was
induced by adding 1 mM isopropyl-1-thio-
-D-galactopyranoside. Cells were
harvested at A600 around 2-2.5 and resuspended
in Buffer A (20 mM sodium phosphate, pH 8.0, 300 mM NaCl) containing 1.5% Triton X-100, 1.5% glycerol, and
1 mg·ml
1 HEWL before sonication. Protein extraction was
also performed in the absence of exogenous lysozyme to obtain
lysozyme-free ykfE protein after the existence of a complex had been
recognized. In both cases, purification was achieved by nickel affinity
chromatography. The cleared lysate was applied to a 5-ml HiTrap
chelating column (Amersham Pharmacia Biotech) charged with
Ni2+ and was washed with 10 column volumes of Buffer
A, followed by 10 column volumes of Buffer A containing 25 mM imidazole, and 5 column volumes of Buffer A containing
70 mM imidazole at a flow rate of 1 ml·min
1. Elution was performed with a linear gradient
over 8 column volumes from 70 to 500 mM imidazole. The
recombinant protein was eluted with 150-200 mM imidazole,
and fractions were pooled and desalted against 20 mM Tris,
pH 8.0, on a fast desalting column HR 10/10 (Amersham Pharmacia
Biotech) at a flow rate of 5 ml·min
1. Protein
concentration was determined by UV absorption at 280 nm using
extinction coefficients calculated on the basis of tyrosine and
tryptophan contents (8). Protein purity was assessed by SDS
polyacrylamide gel electrophoresis and isoelectrofocusing (IEF) using 3 to 10 pH gradient pre-cast gels (Novex). Preliminary molecular weights
for the purified proteins were estimated by gel filtration using a
calibrated Superdex 75 HR10/30 column (Amersham Pharmacia Biotech)
equilibrated with a 20 mM sodium citrate buffer, pH 6.5, at
a flow rate of 0.5 ml·min
1. The purified proteins were
characterized by mass spectroscopy (matrix-assisted laser desorption
ionization/time of flight, Voyager DE-RP; PerSeptive Biosystems)
and by N-terminal Edman sequencing (473A; Applied Biosystems).
Interaction Measurements--
To assay the interaction between
Ivy and HEWL, 50 mg of HEWL were loaded on a nickel column at a flow
rate of 1 ml·min
1 of 20 mM sodium
phosphate, pH 8.0, 300 mM NaCl in the presence or absence
of 3 mg of pure Ivy protein. After extensive washing with 20 mM sodium phosphate, pH 8.0, 1 M NaCl, elution
was performed with a linear gradient over 5 column volumes to 1 M imidazole
Intrinsic protein fluorescence was measured with a Spex Fluorolog3
photon-counting spectrofluorimeter (Jobin Yvon-Spex, Longjumeau, France) equipped with a 450-watt Xenon source and a cooled
photomultiplier. Tryptophan fluorescence emission spectra were recorded
between 290 and 450 nm from solutions containing the individual
proteins and from solutions containing a mixture of proteins excited
with 280 nm of light. The degree of protein-protein interaction was determined from the extent of fluorescence quenching observed at 344 nm
when spectra of a mixture of proteins were compared with the sum of the
individual protein spectra at the same concentration. Interaction-dependent fluorescence quenching was determined
in 10 mM Tris-HCl buffer, pH 7.0, 8.0, or 9.0, containing
100 mM NaCl at protein concentrations varying from 1 µM to 0.5 nM.
Determination of the Apparent Dissociation Constant
(Ki)--
HEWL activity assay was performed at 25 °C in 100 mM potassium phosphate, pH 6.4, using 0.125 mg·ml
1 Micrococcus lysodeikticus
(Sigma) as substrate. Inhibition studies were carried out by
monitoring the change in turbidity associated with the lysis of
M. lysodeikticus cells as described previously (9). One unit
of HEWL activity was defined as the amount of enzyme causing a decrease
in extinction of 0.001 per min at 450 nm. Ki value
was determined according to the slow tight binding competitive
inhibition model (with no conformational change) (10, 11). The
following equation was used,
|
(Eq. 1)
|
where Ki is the apparent dissociation
constant, Et is the total enzyme (HEWL) concentration, It is the total
inhibitor (Ivy) concentration, VIvy is the
inhibited velocity for a given concentration of Ivy, and
V0 is the velocity in the absence of inhibitor.
HEWL (70 nM) was pre-incubated with Ivy (0-200
nM) at room temperature for 15 min prior to the addition of
the M. lysodeikticus substrate (0.125 mg·ml
1). The Ki value was determined
by fitting the experimental data onto the
VIvy/V0 theoretical
curves computed from the above equation (see Fig. 4).
Analysis of the Specificity of Ivy--
The effect of Ivy on
C-type lysozyme activity was determined in the presence of 0.5 µg·ml
1 of HEWL (Sigma), with increasing
concentrations of Ivy from 0 to 100 µg·ml
1. The
effect on
phage lysozyme activity was assayed in 20 mM Tris, pH 8.0, at 25 °C, according to Soumillion et al.
(12) and using chloroform-treated E. coli K-12 MG1655 cells
as substrate. Activity was determined by measuring the decrease of
turbidity over time at 570 nm in the presence of 0.04 µg·ml
1 of
phage lysozyme and concentrations of
Ivy ranging from 0 to 6 µg·ml
1. The chitinase assay
was performed in a 200 mM potassium phosphate buffer, pH
6.0, and 2 mM CaCl2 using crab shell chitin
covalently linked with remazol brilliant violet 5R (Sigma) as
substrate, according to Hackman and Golberg (13). The effect of Ivy on chitinase activity was determined in the presence of 0.5 µg·ml
1 of Streptomyces griseus chitinase
(Fluka) at Ivy concentrations ranging from 0 to 5 µg·ml
1. The enzymatic activity was monitored at 575 nm.
Human saliva was also used as a source of lysozyme. 10 µl of fresh
saliva were mixed with 1 ml of 100 mM potassium phosphate, pH 6.4, containing 0.125 mg·ml
1 M. lysodeikticus. Concentrations of Ivy ranging from 0 to 20 µg·ml
1 were used for this assay.
 |
RESULTS AND DISCUSSION |
Exogenous HEWL is usually added prior to sonication to help
the disruption of the E. coli cell wall according to the
usual extraction protocol for recombinant proteins. HEWL is then
removed during the subsequent purification steps. In the case of Ivy, a
succession of anomalies led us to suspect a strong interaction between
the two proteins. After the purification step by metal chelating
chromatography on a nickel resin, SDS polyacrylamide gel
electrophoresis analyses of the eluted proteins revealed the presence
of two bands of nearly identical molecular mass, around 15 kDa, thus
close to the predicted value for the mature form of Ivy (without signal
peptide). Mass spectrometry and N-terminal sequencing clearly indicated
that these fractions consisted of a mixture of two proteins present in
equivalent quantities and identified one of them as the mature Ivy
protein (molecular mass = 15.04 kDa) and the other as the
exogenous HEWL (molecular mass = 14.3 kDa).
Preliminary results suggested the existence of a specific interaction
between the two proteins. During metal chelating chromatography on a
nickel resin, HEWL could only be retained if Ivy (extracted in the
absence of lysozyme) had first been trapped on the column (see
"Experimental Procedures"). The SDS polyacrylamide gel
electrophoresis analysis of the eluted fractions confirmed the
co-elution of Ivy and HEWL. Finally, the effect of the increase of HEWL
concentrations on the IEF behavior of Ivy also suggested a strong
interaction (Fig. 1). In the absence of
HEWL, the IEF migration of Ivy exhibited two close bands at pI = 7.0 and pI = 6.7 (for a theoretical pI of 6.74). These two bands
most likely correspond to the dimeric and monomeric forms of Ivy in
solution. HEWL alone was found to migrate at pI>10. The increase of
HEWL concentrations resulted in a decrease of intensity of the Ivy
bands at pI = 7.0 and pI = 6.7 (Fig. 1) and the appearance of
an extra band of material reverse migrating into the gel wells, at pI
10.
The HEWL-Ivy interaction was further studied by fluorescence
spectroscopy. The fluorescence spectrum of a 1 µM
lysozyme, 1 µM Ivy mixture was found to differ
significantly from that expected when adding the fluorescence emission
spectra of the individual proteins. An overall 20% quenching of the
fluorescence was measured with maximal quenching at 344 nm (Fig.
2). The shape and maximum of the spectrum
are consistent with at least one relatively exposed tryptophan
being quenched in the lysozyme-Ivy complex. The examination of the
concentration dependence of the quenching spectrum showed that the
shape of this spectrum was independent of the protein concentration
between 1 nM and 1 µM. No reliable
Kd value measurement could be obtained because of
the insufficient intrinsic fluorescence intensity in the nM
concentration range.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
Intrinsic fluorescence quenching because of
HEWL-Ivy interaction. The figure shows the fluorescence emission
spectra of 300 nM HEWL (lower solid curve) and
300 nM Ivy (dashed curve) obtained separately.
The dotted curve shows the experimental spectrum obtained
for a 1:1 mixture of HEWL and Ivy, total protein concentration 600 nM. The upper solid curve is the expected
emission spectrum for the mixture obtained by adding the spectra of the
individual proteins. The difference between the upper solid
and dotted curves (a quenching of ~20%) is attributed to
the interaction between the two proteins. The spectra shown in
this figure were obtained at pH 9.0. AU, arbitrary
unit.
|
|
Finally, co-crystallization experiments and the subsequent analysis of
the crystal content demonstrated the presence of both proteins, thus
suggesting a specific and stable interaction between the two molecules
(data not shown). The determination of the complex three-dimensional
structure is currently in progress.
In addition to its specific physical interaction with HEWL, Ivy is also
a potent inhibitor of HEWL enzymatic activity (Fig. 3). Preliminary experiments showed that
in the presence of 1 µg·ml
1 of Ivy, the addition of 1 µg·ml
1 of HEWL produced a nonlinear kinetic with an
upward concavity (Fig. 3a, curve b). In contrast,
the pre-incubation of Ivy with HEWL for 15 min resulted in kinetic
exhibiting a slight downward concavity (Fig. 3a, curve
c). These results suggest a slow binding kinetic model for the
Ivy-HEWL interaction. In addition, near-complete inhibition is reached
for a range of Ivy concentrations comparable with the concentration of
HEWL (see Figs. 3b and Fig.
4), indicating that Ivy behaves as a slow
tight binding inhibitor. A Ki value of about 1 nM was thus estimated by fitting the experimental data
(Fig. 4) with Morrison's equation corresponding to this model (see Eq. 1 and Refs. 10 and 11).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Progress curves of inhibition of HEWL by
ykfE/Ivy. a, progress of hydrolysis of cell walls of
M. lysodeikticus by 1 µg·ml 1 of HEWL
(curve a) in the presence of 1 µg·ml 1 of
Ivy without pre-incubation (curve b) or with 15-min.
pre-incubation (curve c). The upward concavity of
curve b corresponds to the latency in the inhibitory complex
formation. Conversely, the slight downward concavity in curve
c reflects a small dissociation (Ki 1 nM) of the inhibitory complex because of its initial
dilution (70×) in the substrate mixture. b, progress of
hydrolysis of cell walls of M. lysodeikticus by the addition
of 1 µg·ml 1 of HEWL (curve a) to
increasing concentrations of Ivy as follows: 1.0 (curve b);
1.2 (curve c); 1.6 (curve d), and 3.2 µg·ml 1 (curve e) without
pre-incubation.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of increasing concentrations of
ykfE/Ivy on HEWL activity. The HEWL activity is plotted
(black circles) for increasing Ivy concentrations. HEWL (70 nM) and Ivy (0 to 200 nM) were pre-incubated
for 15 min before starting the reaction by adding M. lysodeikticus as substrate. Activities are expressed as a ratio
between VIvy and V0, the
initial velocities in the presence or absence of inhibitor,
respectively. Ki was determined as the value
allowing the best fit of the experimental data onto a slow competitive
tight binding inhibition model described by Eq. 1 (see text).
The curve corresponds to the model for a
Ki value of 1 nM
|
|
The previous experiments demonstrated the potent inhibitory activity of
Ivy on hen egg white lysozyme. We then explored the effect of Ivy on
the related proteins of increasing evolutionary divergence. We selected
a set of lysozyme and lysozyme-like proteins based on structural
similarity using the MMDB data base (14). Using HEWL as initial query
(MMDB accession number 1151),
phage lysozyme (root mean
square deviation, 1.3 Å; 21.1% identity), and chitinase (root mean
square deviation, 1.9 Å; 11.1% identity) were selected as
representatives of structural homologs with low sequence similarity.
The inhibitory effect of Ivy was thus tested on the two proteins. Ivy
was found to cause a weak inhibition of
phage lysozyme (Fig.
5). The activity was only reduced by 15%
at a molar ratio of 200:1, Ivy:
phage lysozyme. We found no
inhibitory effect of Ivy on chitinase from S. griseus. We
then investigated the capacity of Ivy to inhibit other C-type lysozymes and tested human saliva, because this secretion was reported to contain
30-55 µg·ml
1 of lysozyme (15). Ivy was found to
strongly inhibit the lysozyme activity in saliva (Fig.
6). Around 50 µg·ml
1 of
Ivy is sufficient to observe a decrease of 50% of the activity, which
was fully abolished for an Ivy concentration of 0.5 mg per ml of
saliva.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
Comparative inhibition of HEWL (black
circles) and phage lysozyme
(open circles) activity by increasing concentrations
of ykfE/Ivy.
|
|
On a gel filtration column, Ivy is eluted with an apparent molecular
mass of about 30 kDa, indicating that the predominant form in solution
is a homodimer, as already suggested by IEF experiments. Fluorescence
studies confirmed this model. The fluorescence emission spectrum of
HEWL exhibits a broad peak with a maximum at 342 nm and a long
wavelength tail typical of relatively exposed tryptophan residues. In
contrast, the spectrum of Ivy shows a peak at 334 nm ~25% more
intense on an absolute scale and 2.5 times more intense on a per
tryptophan scale (Fig. 2). Such intense fluorescence and the relatively
short wavelength of maximum emission both argue for tryptophans buried
within apolar environments. Furthermore, the shape of the emission
spectrum was found to be independent of the protein concentration in a
broad 0.5 nM to 1 µM range and appears
insensitive to change in pH between 7.0 and 9.0. The order of magnitude
of the dimerization Kd appeared much lower than
10
9 M, although no precise measurement could
be made in this concentration range. Altogether, these biophysical
results suggest that the Ivy homodimer is the physiologically active unit.
C-type lysozyme is an ancient protein whose origin goes back
about 500 million years (16). It has long been recognized that lysozymes (the family of enzymes hydrolyzing the 1,4-
linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in
the peptidoglycan moiety of bacterial cell walls) are part of a
nonimmunological ancestral bactericidal system in vertebrates. C-type
lysozyme is found in the serum (17-19), in milk (20, 21), in the
digestive tract (22), in the airway (23), and in all mucosal surfaces and secretions (24-31). There is multiple evidence that lysozymes play
a significant role in the control of the host microflora to prevent
infection (32-36). Bacterial C-type lysozyme inhibitors might thus
have emerged to balance the host defense. Indeed, an increase of
anti-lysozyme activity has been linked to bacterial persistence in
several systems (37, 38). However, lysozyme inhibitors could also be
directed against lysozyme activities of other microorganisms and play a
role in ecological competition (39, 40). Finally, these inhibitors
could also have emerged as a protection against bacteriophage-encoded
lysozymes, the activity of which is essential to the release of mature
virions (41). It thus makes evolutionary sense that bacteria might have
evolved a resistance mechanism against the bactericidal activity of
various lysozymes found in their environment. However, it must be noted that for Gram-negative bacteria such as E. coli, the
presence of an outer membrane impermeable to molecules larger than 0.6 kDa should be sufficient to protect the peptidoglycan moiety from the
lysozymes present in the medium.
We selected ykfE as an ORFan gene expressed by
E. coli K12 (5). The presence of a signal peptide predicted
a periplasmic location for its protein product, which is now consistent
with its newly assigned function. Our biochemical and functional
analyses of Ivy show that its predominant homodimeric form strongly
interacts with hen and human, and probably all C-type lysozymes,
thereby abrogating their activity in a stoichiometric manner. Ivy does inhibit C-type lysozymes under physiological conditions, as tested in
human saliva, the secretion where lysozyme is naturally found at the
highest concentration. It is thus likely that at least one purpose of
Ivy is to protect E. coli from its natural host lysozyme
bactericidal activity, for instance in cases where the integrity of the
outer membrane might be compromised (e.g. by chemically aggressive compounds in the medium or at the time of cell division).
As such a resistance mechanism against an ubiquitous bactericidal
enzyme should be advantageous to all murein-containing bacteria, in
particular Gram-positive bacteria, ykfE/Ivy-like genes are expected to exist in many bacterial genomes, making its ORFan nature a
paradox. Indeed, we detected a putative ortholog of E. coli
Ivy within the recently published genome of Pseudomonas
aeruginosa (1). However, the two protein sequences only share 30%
of identical residues in their most similar region, indicating a fast
divergence rate (Fig. 7). It is thus
likely that genes of the ykfE/Ivy family are not detected in
other bacteria because of their low sequence conservation. It is our
hope that the knowledge of the three-dimensional structure of Ivy will
allow the discovery of other Ivy homologues by the
identification of a set of critical positions in the sequence beyond
the twilight zone of sequence similarity.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Amino acid sequence alignment of ykfE/Ivy
versus P. aeruginosa ORF PA3902. The
depicted alignment has been produced using default parameters in FASTA
(42). The underlined sequence corresponds to the known
signal peptide in Ivy.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. C. Cambillau for access to
x-ray diffraction equipment and helpful discussions and
Dr. Mary Berlin for quick validation of the Ivy name. We also
acknowledge the helpful comments of anonymous referees concerning the
analysis of the Ivy inhibition mechanism. We thank Dr. C. Evrard for
the gift of
phage lysozyme.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 33 04 91 16 45 48; Fax: 33 04 91 16 45 49; E-mail:
vincent.monchois@igs.cnrs-mrs.fr.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M010297200
 |
ABBREVIATIONS |
The abbreviations used are:
ORF(s), open reading frame(s);
HEWL, hen egg white lysozyme;
IEF, isoelectrofocusing.
 |
REFERENCES |
1.
|
Stover, C. K.,
Pham, X. Q.,
Erwin, A. L.,
Mizoguchi, S. D.,
Warrener, P.,
Hickey, M. J.,
Brinkman, F. S.,
Hufnagle, W. O.,
Kowalik, D. J.,
Lagrou, M.,
Garber, R. L.,
Goltry, L.,
Tolentino, E.,
Westbrock-Wadman, S.,
Yuan, Y.,
Brody, L. L.,
Coulter, S. N.,
Folger, K. R.,
Kas, A.,
Larbig, K.,
Lim, R.,
Smith, K.,
Spencer, D.,
Wong, G. K.,
Wu, Z.,
and Paulsen, I. T.
(2000)
Nature
406,
959-964[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Fischer, D.,
and Eisenberg, D.
(1999)
Bioinformatics
15,
759-762[Free Full Text]
|
3.
|
Alimi, J. P.,
Poirot, O.,
Lopez, F.,
and Claverie, J. M.
(2000)
Genome Res.
10,
959-966[Abstract/Free Full Text]
|
4.
|
Blattner, F. R.,
Plunkett, G.,
Bloch, C. A.,
Perna, N. T.,
Burland, V.,
Riley, M.,
Collado-Vides, J.,
Glasner, J. D.,
Rode, C. K.,
Mayhew, G. F.,
et al..
(1997)
Science
277,
1453-1474[Abstract/Free Full Text]
|
5.
|
Wasinger, V. C.,
and Humphery-Smith, I.
(1998)
FEMS Microbiol. Lett.
169,
375-382[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Pasquali, C.,
Frutiger, S.,
Wilkins, M. R.,
Hughes, G. J.,
Appel, R. D.,
Bairoch, A.,
Schaller, D.,
Sanchez, J. C.,
and Hochstrasser, D. F.
(1996)
Electrophoresis
17,
547-555[Medline]
[Order article via Infotrieve]
|
7.
|
Abergel, C.,
Monchois, V.,
Chenivesse, S.,
Jeudy, S.,
and Claverie, J. M.
(2000)
Acta Crystallogr.
56,
1694-1695
|
8.
|
Gill, S. C.,
and von Hippel, P. H.
(1989)
Anal. Biochem.
182,
319-326[Medline]
[Order article via Infotrieve]
|
9.
| Charlemagne, D., and Jollès, P. (1970) C. R. Acad.
Sci. (Paris) 2721-2723
|
10.
|
Morrison, J. F.
(1969)
Biochim. Biophys. Acta
185,
269-286[Medline]
[Order article via Infotrieve]
|
11.
|
Morrison, J. F.,
and Walsh, C.
(1988)
Adv. Enzymol. Relat. Areas Mol. Biol.
61,
201-301[Medline]
[Order article via Infotrieve]
|
12.
|
Soumillion, P.,
Jespers, L.,
Vervoors, J.,
and Fastrez, J.
(1995)
Protein Eng.
8,
451-456[Abstract]
|
13.
|
Hackman, R. H.,
and Golberg, M.
(1964)
Anal. Biochem.
8,
397-401
|
14.
|
Wang, Y.,
Addess, K. J.,
Geer, L.,
Madej, T.,
Marchler-Bauer, A.,
Zimmerman, D.,
and Bryant, S. H.
(2000)
Nucleic Acids Res.
28,
243-245[Abstract/Free Full Text]
|
15.
|
Taylor, D. C.,
Cripps, A. W.,
and Clancy, R. L.
(1992)
J. Immunol. Methods
146,
55-61[Medline]
[Order article via Infotrieve]
|
16.
|
Qasba, P. K.,
and Kumar, S.
(1997)
Crit. Rev. Biochem. Mol. Biol.
32,
255-306[Abstract]
|
17.
|
Wilson, K. P.,
Malcom, B. A.,
and Matthews, B. W.
(1992)
J. Biol. Chem.
267,
10842-10849[Abstract/Free Full Text]
|
18.
|
Gemsa, D.,
Davis, S. D.,
and Wedgwood, R. J.
(1996)
Nature
210,
950-951
|
19.
|
Selsted, M. E.,
and Martinez, R. J.
(1978)
Infect. Immun.
20,
782-791[Medline]
[Order article via Infotrieve]
|
20.
|
Murakami, K.,
Lagarde, M.,
and Yuki, Y.
(1998)
Electrophoresis
19,
2521-2527[Medline]
[Order article via Infotrieve]
|
21.
|
Hamosh, M.
(1998)
Biol. Neonate
74,
163-176[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Peeters, T.,
and Vantrappen, G.
(1975)
Gut
16,
553-558[Abstract]
|
23.
|
Jacquot, J.,
Hayem, A.,
and Galabert, C.
(1992)
Eur. Respir. J.
5,
343-358[Abstract]
|
24.
|
Lim, D. J.,
Liu, Y. S.,
and Birck, H.
(1976)
Ann. Otol. Rhinol. Laryngol.
85,
50-60[Medline]
[Order article via Infotrieve]
|
25.
|
McNabb, P. C.,
and Tomasi, T. B.
(1981)
Annu. Rev. Microbiol.
35,
477-496[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Cohen, M. S.,
Black, J. R.,
Proctor, R. A.,
and Sparling, P. F.
(1984)
Scand. J. Urol. Nephrol. Suppl.
86,
13-22[Medline]
[Order article via Infotrieve]
|
27.
|
Tachibana, M.,
Morioka, H.,
Machino, M.,
and Mizukoshi, O.
(1986)
Auris Nasus Larynx
13,
97-99[Medline]
[Order article via Infotrieve]
|
28.
|
Hanamure, Y.,
and Lim, D. J.
(1986)
Am. J. Otolaryngol.
7,
410-425[Medline]
[Order article via Infotrieve]
|
29.
|
McClellan, K. A.
(1997)
Surv. Ophthalmol.
42,
233-246[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Schenkels, L. C.,
Veerman, E. C.,
and Nieuw Amerongen, A. V.
(1995)
Crit. Rev. Oral Biol. Med.
6,
161-175[Abstract]
|
31.
|
Cole, A. M.,
Dewan, P.,
and Ganz, T.
(1999)
Infect. Immun.
67,
3267-3275[Abstract/Free Full Text]
|
32.
|
Kondo, L. R.,
Hanna, L.,
and Keshishyan, H.
(1973)
Proc. Soc. Exp. Biol. Med.
142,
131-132
|
33.
|
Walker, W. A.
(1979)
Ciba Found. Symp.
70,
201-219[Medline]
[Order article via Infotrieve]
|
34.
|
Ved'mina, E. A.,
Pasternak, N. A.,
Shenderovich, V. A.,
Zhuravleva, T. P.,
and Andrusenko, I. T.
(1979)
Antibiot. Khimioter.
24,
746-750
|
35.
|
Hancock, R. E.,
and Scott, M. G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8856-8861[Abstract/Free Full Text]
|
36.
|
Vollmer, W.,
and Tomasz, A.
(2000)
J. Biol. Chem.
275,
20496-20501[Abstract/Free Full Text]
|
37.
|
Bukharin, O. V.
(1994)
Zh. Mikrobiol. Epidemiol. Immunobiol.
1, (Suppl.),
4-13
|
38.
|
Bondarenko, V. M.,
Petrovskaia, V. G.,
and Iablochkov, A. L.
(1994)
Zh. Mikrobiol. Epidemiol. Immunobiol.
1, (Suppl.),
22-28
|
39.
|
Nemtseva, N. V.
(1997)
Zh. Mikrobiol. Epidemiol. Immunobiol.
4,
123-126[Medline]
[Order article via Infotrieve]
|
40.
|
Lentsner, A. A.,
Lentsner, Kh. P.,
and Toom, M. A.
(1975)
Zh. Mikrobiol. Epidemiol. Immunobiol.
8,
77-81[Medline]
[Order article via Infotrieve]
|
41.
|
Bukharin, O. V.,
and Deriabin, D. G.
(1989)
Zh. Mikrobiol. Epidemiol. Immunobiol.
11,
16-19[Medline]
[Order article via Infotrieve]
|
42.
|
Pearson, W. R.
(2000)
Methods Mol. Biol.
132,
185-219[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.