U.S. patent application number 17/491845 was filed with the patent office on 2022-01-20 for lysin agent and method of use for diagnostic testing.
This patent application is currently assigned to The Government of the United States of America, as represented by the Secretary of Homeland Security. The applicant listed for this patent is The Government of the United States of America, as represented by the Secretary of Homeland Security, The Government of the United States of America, as represented by the Secretary of Homeland Security. Invention is credited to David R. Hodge, Segaran Pillai, Bernard Quigley, Linda Weigel.
Application Number | 20220017884 17/491845 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220017884 |
Kind Code |
A1 |
Pillai; Segaran ; et
al. |
January 20, 2022 |
Lysin Agent and Method of Use for Diagnostic Testing
Abstract
Novel Bacillus lysin proteins have been identified and
characterized. Methods to detect the presence or absence of
bacteria in a sample, methods for lysing bacteria, and methods for
decontaminating or disinfecting areas contaminated with bacteria
that utilize these lysin proteins have also been developed.
Inventors: |
Pillai; Segaran; (Laurel,
MD) ; Weigel; Linda; (Decatur, GA) ; Quigley;
Bernard; (Atlanta, GA) ; Hodge; David R.;
(Washington, DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America, as represented by
the Secretary of Homeland Security |
Washington |
DC |
US |
|
|
Assignee: |
The Government of the United States
of America, as represented by the Secretary of Homeland
Security
Washington
DC
|
Appl. No.: |
17/491845 |
Filed: |
October 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16262258 |
Jan 30, 2019 |
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17491845 |
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15047319 |
Feb 18, 2016 |
10214732 |
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16262258 |
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International
Class: |
C12N 9/24 20060101
C12N009/24; C12Q 1/689 20060101 C12Q001/689; A61K 38/47 20060101
A61K038/47; A61K 38/12 20060101 A61K038/12; A01N 47/44 20060101
A01N047/44 |
Goverment Interests
GOVERNMENT INTEREST
[0002] The subject matter of this disclosure was made with support
from the United States Department of Homeland Security (DHS). The
Government of the United States of America has certain rights in
this invention.
Claims
1-22. (canceled)
23. A method for selecting a candidate lysin polypeptide for
molecular diagnostic testing comprising: (a) culturing a host cell
comprising a vector comprising a nucleic acid sequence encoding the
candidate lysin polypeptide, wherein the nucleic acid sequence is
from a library of polynucleotide fragments; (b) expressing the
candidate lysin polypeptide under conditions in which a nucleic
acid molecule encoding the candidate lysin polypeptide is
expressed; (c) isolating the candidate lysin polypeptide; (d)
incubating the candidate lysin polypeptide with a bacteria; and (e)
selecting the nucleic acid sequence which encodes the candidate
lysin polypeptide which exhibits peptidoglycan hydrolase activity
against the bacteria.
24. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 50% identity
with a nucleic acid sequence of a model lytic enzyme.
25. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 50% similarity
with a nucleic acid sequence of a model lytic enzyme.
26. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 60% identity
with a nucleic acid sequence of a model lytic enzyme.
27. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 60% similarity
with a nucleic acid sequence of a model lytic enzyme.
28. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having between 70%-90%
identity with a nucleic acid sequence of a model lytic enzyme.
29. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having between 70%-90%
similarity with a nucleic acid sequence of a model lytic
enzyme.
30. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 90% identity
with a nucleic acid sequence of a model lytic enzyme.
31. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 90% similarity
with a nucleic acid sequence of a model lytic enzyme.
32. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 95% identity
with a nucleic acid sequence of a model lytic enzyme.
33. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 95% similarity
with a nucleic acid sequence of a model lytic enzyme.
34. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 50% identity with an amino acid sequence of a model lytic
enzyme.
35. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 50% similarity with an amino acid sequence of a model
lytic enzyme.
36. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 60% identity with an amino acid sequence of a model lytic
enzyme.
37. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 60% similarity with an amino acid sequence of a model
lytic enzyme.
38. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
between 70%-90% similarity with an amino acid sequence of a model
lytic enzyme.
39. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
between 70%-90% identity with an amino acid sequence of a model
lytic enzyme.
40. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 90% identity with an amino acid sequence of a model lytic
enzyme.
41. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 90% similarity with an amino acid sequence of a model
lytic enzyme.
42. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 95% identity with an amino acid sequence of a model lytic
enzyme.
43. The method of claim 23, wherein the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 95% similarity with an amino acid sequence of a model
lytic enzyme.
44. The method of claim 23, wherein the bacteria is a gram-positive
bacteria.
45. The method of claim 44, wherein the gram-positive bacteria is a
Bacillus species.
46. The method of claim 45, wherein the Bacillus species is
Bacillus anthracis.
47. The method of claim 23, wherein the bacteria is a gram-negative
bacteria.
48. The method of claim 47, wherein the gram-negative bacteria is a
Yersinia species.
49. The method of claim 48, wherein the Yersinia species is
Yersinia pestis.
50. A method for decontaminating a surface or room or area or
object contaminated with bacteria comprising contacting the surface
or room or area or object with a composition comprising an isolated
polypeptide having peptidoglycan hydrolase activity against the
bacteria, wherein the isolated polypeptide comprises the amino acid
sequence of SEQ ID NO: 2, or a fusion thereof.
51. The method of claim 50, wherein the bacteria is a gram-positive
bacteria.
52. The method of claim 51, wherein the gram-positive bacteria is a
Bacillus species.
53. The method of claim 52, wherein the Bacillus species is
Bacillus anthracis.
54. The method of claim 50, wherein the bacteria is a gram-negative
bacteria.
55. The method of claim 54, wherein the gram-negative bacteria is a
Yersinia species.
56. The method of claim 55, wherein the Yersinia species is
Yersinia pestis.
57. A method for disinfecting a surface or room or area or object
contaminated with bacteria comprising contacting the surface or
room or area or object with a composition comprising an isolated
polypeptide having peptidoglycan hydrolase activity against the
bacteria, wherein the isolated polypeptide comprises the amino acid
sequence of SEQ ID NO: 2, or a fusion thereof.
58. The method of claim 57, wherein the bacteria is a gram-positive
bacteria.
59. The method of claim 58, wherein the gram-positive bacteria is a
Bacillus species.
60. The method of claim 59, wherein the Bacillus species is
Bacillus anthracis.
61. The method of claim 57, wherein the bacteria is a gram-negative
bacteria.
62. The method of claim 61, wherein the gram-negative bacteria is a
Yersinia species.
63. The method of claim 62, wherein the Yersinia species is
Yersinia pestis.
64. A method for treating a bacterial infection comprising
administering to a subject in need of such treatment, a composition
comprising an antibiotic that is effective against the bacteria and
an isolated polypeptide having peptidoglycan hydrolase activity
against the bacteria, wherein the polypeptide comprises the amino
acid sequence of SEQ ID NO: 2, or a fusion thereof.
65. The method of claim 64, wherein the bacteria is a gram-positive
bacteria.
66. The method of claim 65, wherein the gram-positive bacteria is a
Bacillus species.
67. The method of claim 66, wherein the Bacillus species is
Bacillus anthracis.
68. The method of claim 64, wherein the bacteria is a gram-negative
bacteria.
69. The method of claim 68, wherein the gram-negative bacteria is a
Yersinia species.
70. The method of claim 69, wherein the Yersinia species is
Yersinia pestis.
71. The method of claim 64, wherein the antibiotic is
polymyxin.
72. The method of claim 71, wherein the polymyxin is administered
at a concentration that is non-toxic to the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
15/047,319 filed Feb. 18, 2016, which is herein incorporated by
reference in its entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. The ASCII copy is named
DHS-056US01 Sequence Listing.txt and is 23 kb in size.
FIELD OF THE DISCLOSURE
[0004] The present disclosure relates to methods and compositions
useful for identification of lytic enzymes. The disclosure relates
to one or more novel peptidoglycan hydrolases that preferentially
or specifically attacks the peptidoglycan cell wall of bacteria,
and the use of the hydrolases as a lytic agent independently or in
conjunction with molecular diagnostic testing for the rapid
identification and lysing of Bacillus anthracis and certain related
bacteria.
BACKGROUND OF THE INVENTION
[0005] A bacterial cell wall is a highly complex structure that
encases the cell, provides it shape and maintains internal osmotic
forces. The cell wall includes a thick layer of peptidoglycan,
which is a heteropolysaccharide polymer comprised of subunits of
alternating N-acetylglucosamine (Glc-NAc) and N-acetylmuramic
(MurNAc) acid (also known as the glycan chain). The MurNAc moiety
has a pentapeptide chain attached. Included subunits are linked by
(31-4 glycosidic bonds and cross-linked via the peptide chains
through alternating L- and D-amino acids. Various types of
peptidoglycan are identified by the type of cross-linkage and the
specific amino acid at the third position of the peptide chain.
Bacillus anthracis has an Aly type peptidoglycan, with the "A"
indicating cross-linkage between positions 3 and 4 in the peptide
chain; "1" indicating that it is a direct cross-linkage; and the
".gamma." (gamma) indicating that the direct cross-linkage is
between meso diaminopimelic acid residue (DAP) and the D-Ala in
position 4. Recent studies (such as Candela, T. et al.;
N-Acetylglucosamine Deacetylases Module the Anchoring of the
Gamma-Glutamyl Capsule to the Cell Wall of Bacillus Anthracis.
Microbial Drug Resistance. 2014, 20, 222-230) indicate a high level
(e.g., 92%) of peptidoglycan modification by N-deacylation of
GlcNAc residues. This modification is typically responsible for the
cell wall's resistance to lytic enzymes such as lysozyme and
mutanolysin. Candela, T. et al., reported that the cross-linkage of
peptidoglycan in Bacillus anthracis is higher than previously
reported.
[0006] Peptidoglycan hydrolases are a diverse group of enzymes that
cleave the cell wall at specific structural sites and are
responsible for the highly-regulated cleavage of peptidoglycan
during cell growth and division. Peptidoglycan hydrolases can be
organized into 4 classes: (i) amidases, (ii) endopeptidases, (iii)
glucosaminidases, and (iv) lysozymes, each of which cleaves a
specific bond within the peptidoglycan or its fragments.
Peptidoglycan hydrolases typically comprise a substrate binding
domain and an activity domain. The activity of a particular
peptidoglycan hydrolase (lysin) may be dependent on one or both of
these domains. Exogenous application of some lysin proteins to
bacterial cells results in hydrolysis of peptidoglycan, and cell
lysis due to osmotic shock.
SUMMARY OF THE INVENTION
[0007] In one or more embodiments, novel peptidoglycan hydrolase
lytic enzymes are described. A vector comprising the nucleic acid
molecule having of SEQ ID NO: 1 is also described in one or more
embodiments. One or more embodiments of the disclosure include an
isolated, and/or purified nucleic acid molecule encoding a
polypeptide having peptidoglycan hydrolase activity, a fragment or
variant or derivative thereof or a fusion of the polypeptide,
fragment, variant, or derivative, a host cell containing a nucleic
acid molecule or a vector. One or more diagnostic substances
including one or more polypeptides, fragments or variants or
derivatives thereof, or one or more fusions of the polypeptide,
fragment, variant, or derivative, are also described. One or more
diagnostic assays including a polypeptide having peptidoglycan
hydrolase activity, a fragment or variant, or derivative thereof or
a fusion of the polypeptide, fragment, variant, or derivative are
also described.
[0008] In one or more embodiments, the present disclosure
describes: (1) creating an in-silico method to identify lysin(s)
with predicted peptidoglycan hydrolytic activity against an intact
cell wall of Bacillus anthracis. In one or more embodiments,
methods to identify potential Bacillus anthracis lysin candidates
by sequence similarity to known lysin proteins from bacteriophage,
prokaryotic (including, but not limited to gram positive or gram
negative), and eukaryotic organisms, and to screen the identified
lysins for usage conditions are described. One or more lytic
peptides having high activity against the intact Bacillus anthracis
cell wall through the design and implementation of a turbidity
reduction assay are described. One or more methods of purifying
lysin proteins through an epitope tag, demonstrating the activity
of the purified protein against whole cells of Bacillus anthracis
is also described.
[0009] The present disclosure further describes one of more
embodiments of the use of a Bacillus anthracis peptidoglycan
hydrolase as a Bacillus anthracis lytic agent independently or in
conjunction with molecular diagnostic tests intended for the rapid
identification of Bacillus anthracis.
[0010] One or more methods are described for selecting a lysin
agent for use in molecular diagnostic testing, including analyzing
genome databases for Bacillus anthracis and near neighbors,
selecting candidate genes encoding potential lytic enzymes based on
conserved amino acid motifs as determined in peptidoglycan
hydrolases, cloning the candidate genes in an expression vector,
isolating proteins thereof, and testing the isolated protein for
lytic activity against Bacillus anthracis, and selecting among the
candidate genes for those which encode proteins with demonstrated
lytic activity for further optimization of lysis conditions. In one
or more embodiments, a vector construct comprising the sequence of
SEQ ID NO: 1 and methods for making the lysin polypeptide BQ22 are
also described.
[0011] In one or more embodiments, this disclosure further
describes isolating and/or purifying (e.g., substantially purified,
sufficiently pure for use in lysing cells) a DNA molecule
comprising a nucleotide sequence encoding a polypeptide comprising
the amino acid sequence of SEQ ID NO: 2; the polypeptide has
peptidoglycan hydrolase activity.
[0012] In one or more embodiments, the present disclosure is
further directed to a method for producing a polypeptide designated
as BQ22 having peptidoglycan hydrolase activity. In one more
embodiments, the method can be comprised of: introducing into an
expression vector a nucleotide sequence comprising the sequence of
SEQ ID NO: 1, the resulting recombinant vector comprising a
nucleotide sequence encoding a protein having the amino acid
sequence of SEQ ID NO: 2, the nucleotide sequence operably linked
to at least one DNA sequence that controls expression of the BQ22
protein. In one or more embodiments, the recombinant vector is
transformed into a host cell, thereby forming a recombinant host
cell; the recombinant host cell is cultured under conditions
suitable for expression of the lysin protein from the DNA molecule
encoding the protein, such that BQ22 polypeptide is produced and
the BQ22 polypeptide is isolated as a purified protein. In one or
more embodiments, the recited order is the order in which the
method is performed.
[0013] In one or embodiments, the present disclosure is further
directed to a method for decontaminating and/or disinfecting a
surface or area or object contaminated with Bacillus anthracis and
certain related bacteria comprising contacting the surface or area
or object with isolated polypeptide, fragment or variant, or
derivative thereof or a fusion of the polypeptide, fragment,
variant, or derivative having peptidoglycan hydrolase activity.
[0014] According to a first aspect, the present disclosure provides
a recombinant nucleic acid comprising a nucleotide sequence
encoding a polypeptide comprising the amino acid sequence of SEQ ID
NO: 2, or a fragment, variant, or derivative thereof or a fusion of
the polypeptide, fragment, variant, or derivative.
[0015] In one or more embodiments, the nucleotide sequence of the
recombinant nucleic acid is operably linked to one or more control
sequences.
[0016] In one or more embodiments, the one or more control
sequences is selected from a group consisting of: a promoter, a
transcriptional start signal, a transcriptional stop signal, a
translational start signal, and a translational stop signal.
[0017] In one or more embodiments, the polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative or fusion thereof comprises a polypeptide tag.
[0018] In one or more embodiments, the polypeptide tag is a
poly-histidine tag.
[0019] In one or more embodiments, the poly-histidine tag is a
hexa-histidine tag.
[0020] In one or more embodiments, the nucleotide sequence encoding
a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
or a fragment, variant, or derivative thereof or a fusion of the
polypeptide, fragment, variant, or derivative, comprises SEQ ID NO:
1.
[0021] According to a second aspect, the present disclosure
provides a vector comprising a recombinant nucleic acid comprising
a nucleotide sequence encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, or a fragment, variant, derivative
thereof or a fusion of the polypeptide, fragment, variant, or
derivative.
[0022] In one or more embodiments, the nucleotide sequence of the
recombinant nucleic acid is operably linked to one or more control
sequences.
[0023] In one or more embodiments, the one or more control
sequences is selected from a group consisting of: a promoter, a
transcriptional start signal, a transcriptional stop signal, a
translational start signal, and a translational stop signal.
[0024] In one or more embodiments, the polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative or fusion thereof comprises a polypeptide tag.
[0025] In one or more embodiments, the polypeptide tag is a
poly-histidine tag.
[0026] In one or more embodiments, the poly-histidine tag is a
hexa-histidine tag.
[0027] In one or more embodiments, the nucleotide sequence encoding
a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
or a fragment, variant, or derivative thereof or a fusion of the
polypeptide, fragment, variant, or derivative, comprises SEQ ID NO:
1.
[0028] According to a third aspect, the present disclosure provides
a host cell transformed with a vector comprising a recombinant
nucleic acid comprising a nucleotide sequence encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or
a fragment, variant, derivative thereof or a fusion of the
polypeptide, fragment, variant, or derivative.
[0029] In one or more embodiments, the nucleotide sequence of the
recombinant nucleic acid is operably linked to one or more control
sequences.
[0030] In one or more embodiments, the one or more control
sequences is selected from a group consisting of: a promoter, a
transcriptional start signal, a transcriptional stop signal, a
translational start signal, and a translational stop signal.
[0031] In one or more embodiments, the polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative or fusion thereof comprises a polypeptide tag.
[0032] In one or more embodiments, the polypeptide tag is a
poly-histidine tag.
[0033] In one or more embodiments, the poly-histidine tag is a
hexa-histidine tag.
[0034] In one or more embodiments, the nucleotide sequence encoding
a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
or a fragment, variant, or derivative thereof or a fusion of the
polypeptide, fragment, variant, or derivative, comprises SEQ ID NO:
1.
[0035] According to a fourth aspect, the present disclosure
provides for an isolated polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, or a fragment, variant, derivative
thereof or a fusion of the polypeptide, fragment, variant, or
derivative, the polypeptide having peptidoglycan hydrolase
activity
[0036] According to a fifth aspect, the present disclosure provides
a method for producing a polypeptide with peptidoglycan hydrolase
activity, comprising (a) providing a host cell comprising a vector
comprising a nucleotide sequence encoding the polypeptide; and (b)
culturing the host cell under culturing conditions; and (c)
isolating the polypeptide, wherein the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2.
[0037] In one or more embodiments, the nucleotide sequence of the
recombinant nucleic acid is operably linked to one or more control
sequences.
[0038] In one or more embodiments, the one or more control
sequences is selected from a group consisting of: a promoter, a
transcriptional start signal, a transcriptional stop signal, a
translational start signal, and a translational stop signal.
[0039] In one or more embodiments, the polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative or fusion thereof comprises a polypeptide tag.
[0040] In one or more embodiments, the polypeptide tag is a
poly-histidine tag.
[0041] In one or more embodiments, the poly-histidine tag is a
hexa-histidine tag.
[0042] In one or more embodiments, the nucleotide sequence encoding
a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
or a fragment, variant, or derivative thereof or a fusion of the
polypeptide, fragment, variant, or derivative, comprises SEQ ID NO:
1.
[0043] According to a sixth aspect, the present disclosure provides
a method for selecting a candidate lysin polypeptide for molecular
diagnostic testing comprising (a) culturing a host cell comprising
a vector comprising a nucleic acid sequence encoding the candidate
lysin polypeptide, wherein the nucleic acid sequence is from a
library of polynucleotide fragments; and (b) expressing the
candidate lysin polypeptide under conditions in which a nucleic
acid molecule encoding the candidate lysin polypeptide is
expressed; and (c) isolating the candidate lysin polypeptide; and
(d) incubating the candidate lysin polypeptide with a bacteria; and
(e) selecting the nucleic acid sequence which encodes the candidate
lysin polypeptide which exhibits peptidoglycan hydrolase activity
against the bacteria.
[0044] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 50% identity
with a nucleic acid sequence of a model lytic enzyme.
[0045] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 50% similarity
with a nucleic acid sequence of a model lytic enzyme.
[0046] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 60% identity
with a nucleic acid sequence of a model lytic enzyme.
[0047] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 60% similarity
with a nucleic acid sequence of a model lytic enzyme.
[0048] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having between 70%-90%
identity with a nucleic acid sequence of a model lytic enzyme.
[0049] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having between 70%-90%
similarity with a nucleic acid sequence of a model lytic
enzyme.
[0050] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 90% identity
with a nucleic acid sequence of a model lytic enzyme.
[0051] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 90% similarity
with a nucleic acid sequence of a model lytic enzyme.
[0052] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 95% identity
with a nucleic acid sequence of a model lytic enzyme.
[0053] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for nucleic acid sequences having at least 95% similarity
with a nucleic acid sequence of a model lytic enzyme.
[0054] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 50% identity with an amino acid sequence of a model lytic
enzyme.
[0055] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 50% similarity with an amino acid sequence of a model
lytic enzyme.
[0056] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 60% identity with an amino acid sequence of a model lytic
enzyme.
[0057] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 60% similarity with an amino acid sequence of a model
lytic enzyme.
[0058] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
between 70%-90% similarity with an amino acid sequence of a model
lytic enzyme.
[0059] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
between 70%-90% identity with an amino acid sequence of a model
lytic enzyme.
[0060] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 90% identity with an amino acid sequence of a model lytic
enzyme.
[0061] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 90% similarity with an amino acid sequence of a model
lytic enzyme.
[0062] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 95% identity with an amino acid sequence of a model lytic
enzyme.
[0063] In one or more embodiments, the library of polynucleotides
is derived from sequences identified in a genome database by
searching for amino acid sequences encoded by the sequences having
at least 95% similarity with an amino acid sequence of a model
lytic enzyme.
[0064] In one or more embodiments, the bacteria is prokaryote.
[0065] In one or more embodiments, the prokaryote is a
gram-positive bacteria.
[0066] In one or more embodiments, the gram-positive bacteria is a
Bacillus species.
[0067] In one or more embodiments, the Bacillus species is Bacillus
anthracis.
[0068] In one or more embodiments, the prokaryote is a
gram-negative bacteria.
[0069] In one or more embodiments, the gram-negative bacteria is a
Yersinia species.
[0070] In one or more embodiments, the Yersinia species is Yersinia
pestis.
[0071] According to a seventh aspect, the present disclosure
provides a method for diagnosing the presence or absence of
bacteria in a sample comprising (a) providing a sample of the
bacteria; and (b) incubating the sample of the bacteria in the
presence of an isolated polypeptide having peptidoglycan hydrolase
activity, and (c) contacting the sample and the isolated
polypeptide having peptidoglycan hydrolase activity with at least a
first and a second oligonucleotide primer under conditions
sufficient to provide polymerase-based nucleic acid amplification;
and (d) detecting In one or more embodiments, the bacteria is
prokaryote.
[0072] In one or more embodiments, the bacteria is prokaryote.
[0073] In one or more embodiments, the prokaryote is a
gram-positive bacteria.
[0074] In one or more embodiments, the gram-positive bacteria is a
Bacillus species.
[0075] In one or more embodiments, the Bacillus species is Bacillus
anthracis.
[0076] In one or more embodiments, the prokaryote is a
gram-negative bacteria.
[0077] In one or more embodiments, the gram-negative bacteria is a
Yersinia species.
[0078] In one or more embodiments, the Yersinia species is Yersinia
pestis.
[0079] In one or more embodiments, the polymerase-based nucleic
acid amplification is quantitative polymerase chain reaction
(QPCR).
[0080] In one or more embodiments, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative thereof, or a fusion of the polypeptide, fragment,
variant, or derivative.
[0081] According to an eighth aspect, the present disclosure
provides a method for lysing bacteria comprising (a) providing a
sample of the bacteria; and (b) preparing an isolated polypeptide
having peptidoglycan hydrolase activity; and (c) incubating the
isolated polypeptide with the bacteria.
[0082] In one or more embodiments, the bacteria is prokaryote.
[0083] In one or more embodiments, the prokaryote is a
gram-positive bacteria.
[0084] In one or more embodiments, the gram-positive bacteria is a
Bacillus species.
[0085] In one or more embodiments, the Bacillus species is Bacillus
anthracis.
[0086] In one or more embodiments, the prokaryote is a
gram-negative bacteria.
[0087] In one or more embodiments, the gram-negative bacteria is a
Yersinia species.
[0088] In one or more embodiments, the Yersinia species is Yersinia
pestis.
[0089] In one or more embodiments, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative thereof, or a fusion of the polypeptide, fragment,
variant, or derivative.
[0090] According to an ninth aspect, the present disclosure
provides a method for decontaminating a surface or room or area or
object contaminated with bacteria comprising contacting the surface
or room or area or object with a composition comprising an isolated
polypeptide having peptidoglycan hydrolase activity against the
bacteria.
[0091] In one or more embodiments, the bacteria is prokaryote.
[0092] In one or more embodiments, the prokaryote is a
gram-positive bacteria.
[0093] In one or more embodiments, the gram-positive bacteria is a
Bacillus species.
[0094] In one or more embodiments, the Bacillus species is Bacillus
anthracis.
[0095] In one or more embodiments, the prokaryote is a
gram-negative bacteria.
[0096] In one or more embodiments, the gram-negative bacteria is a
Yersinia species.
[0097] In one or more embodiments, the Yersinia species is Yersinia
pestis.
[0098] In one or more embodiments, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative thereof, or a fusion of the polypeptide, fragment,
variant, or derivative.
[0099] According to an tenth aspect, the present disclosure
provides a method for disinfecting a surface or room or area or
object contaminated with bacteria comprising contacting the surface
or room or area or object with a composition comprising an isolated
polypeptide having peptidoglycan hydrolase activity against the
bacteria.
[0100] In one or more embodiments, the bacteria is prokaryote.
[0101] In one or more embodiments, the prokaryote is a
gram-positive bacteria.
[0102] In one or more embodiments, the gram-positive bacteria is a
Bacillus species.
[0103] In one or more embodiments, the Bacillus species is Bacillus
anthracis.
[0104] In one or more embodiments, the prokaryote is a
gram-negative bacteria.
[0105] In one or more embodiments, the gram-negative bacteria is a
Yersinia species.
[0106] In one or more embodiments, the Yersinia species is Yersinia
pestis.
[0107] In one or more embodiments, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative thereof, or a fusion of the polypeptide, fragment,
variant, or derivative.
[0108] According to an eleventh aspect, the present disclosure
provides a method for treating a bacterial infection comprising
administering to a subject in need of such treatment, a composition
comprising an antibiotic that is effective against the bacteria and
an isolated polypeptide having peptidoglycan hydrolase activity
against the bacteria.
[0109] In one or more embodiments, the bacteria is prokaryote.
[0110] In one or more embodiments, the prokaryote is a
gram-positive bacteria.
[0111] In one or more embodiments, the gram-positive bacteria is a
Bacillus species.
[0112] In one or more embodiments, the Bacillus species is Bacillus
anthracis.
[0113] In one or more embodiments, the prokaryote is a
gram-negative bacteria.
[0114] In one or more embodiments, the gram-negative bacteria is a
Yersinia species.
[0115] In one or more embodiments, the Yersinia species is Yersinia
pestis.
[0116] In one or more embodiments, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, or a fragment, variant,
derivative thereof, or a fusion of the polypeptide, fragment,
variant, or derivative.
[0117] In one or more embodiments, the antibiotic is polymyxin.
[0118] In one or more embodiments, the polymyxin is administered at
a concentration that is non-toxic to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0119] The accompanying drawings illustrate one or more embodiments
of the present disclosure and, together with the detailed
description and examples sections, serve to explain the principles
and implementations of the disclosure. While the foregoing
disclosure describes one or more embodiments of the invention, it
should be clearly understood that the disclosure is by way of
illustration and example only, and is not limiting.
[0120] FIG. 1 is a diagrammatic view showing the steps in a rapid
antimicrobial susceptibility assay based on the use of one or more
embodiments.
[0121] FIG. 2 is a flow diagram showing a method of finding and
selecting a potential lysin agent for use in molecular diagnostic
testing according to one or more embodiments.
[0122] FIG. 3 illustrates a SDS-PAGE of proteins in whole cell
lysates from IPTG-induced BL21 strains.
[0123] FIG. 4 illustrates a SDS-PAGE gradient gel of the Triton
X-100 (Sigma Aldrich Co., St. Louis, Mo.) soluble protein fractions
from IPTG-induced BL21 Strains.
[0124] FIG. 5 is a graph illustrating lytic activity over a 30 min
time period when Bacillus anthracis vegetative cells are exposed to
crude E. coli lysates that contain the expressed lysin protein.
[0125] FIG. 6 illustrates a 4%-15% SDS-PAGE gradient gel stained to
show IMAC-purified BQ22 (BA_2528 N-terminal 6.times.His), BQ23
proteins (BA_2528 C-terminal 6.times.His), and BCZK2532 fusion
constructs.
[0126] FIG. 7 is a graph illustrating lytic activity of IMAC
purified BQ22 and BQ23 against Bacillus anthracis Sterne.
[0127] FIG. 8 is a graph illustrating activity of IMAC purified
BQ22 is against EDTA-treated Y. pestis.
[0128] FIG. 9 illustrates the DNA sequence features of a vector
construct for BQ22.
DETAILED DESCRIPTION
[0129] The following terminology is provided for convenience and
should not be taken to imply that the terminology is being
redefined herein to be restricted to any specific characteristics,
features, or aspects of the invention with which that terminology
is associated.
[0130] For purposes of the present disclosure,
"peptidoglycan"--refers to a heteropolymer consisting of
N-acetylglucosamine and N-acetylmuramic acid subunits linked by
(31-4 glycosidic bonds and cross-linked by short peptides, forming
a cell wall, which generally surrounds the cell, maintains cell
shape, protects the cell from rupture due to internal turgor
pressure, and serves as a platform for attachment of various
proteins.
[0131] For purposes of the present disclosure, "lysin" refers to an
enzyme that has the ability to hydrolyze bacterial cell walls and
exhibits peptidoglycan hydrolytic activity. Lysins may optionally
include at least one enzymatically active domain (EAD) having at
least one of the following activities: amidases, muramidases,
glucosaminidases, and endopeptidases. In addition, the lysins may
also optionally include regions that are enzymatically inactive,
including, but not limited to cell wall-binding domains (CBDs),
which may serve to bind to the bacterial cell wall. Lysins may
optionally contain two or more CBDs. Lysins also may optionally
refer to enzymes with at least one enzymatically active domain but
no CBD. Generally, cell wall-binding domains bind to specific
components on the cross-linked peptidoglycan heteropolymer that
form the bacterial cell wall. In one or more non-limiting examples,
the cell wall binding domain is a peptidoglycan binding domain and
binds to the bacteria's peptidoglycan structure. In one or more
non-limiting examples, an amino acid linker region may optionally
be present to connect the lysin's different domains.
[0132] For purposes of the present disclosure, "amidase" refers to
any peptidoglycan hydrolytic enzyme that hydrolyzes the muramic
acid-D-alanine bond linking the glycan strand to the peptide
subunit.
[0133] For purposes of the present disclosure, "lysozyme" refers to
any peptidoglycan hydrolytic enzyme that hydrolyzes the .beta.1-4
glycosidic bond between N-acetylmuramic acid and
N-acetylglucosamine.
[0134] For purposes of the present disclosure, "transglycosylase"
refers any peptidoglycan hydrolytic enzyme that hydrolyzes the
.beta.1-4 glycosidic bond between N-acetylmuramic acid and
N-acetylglucosamine resulting in the formation of an
anhydromuramoyl residue.
[0135] For purposes of the present disclosure, "glucosaminidase"
refers to any peptidoglycan hydrolytic enzyme that hydrolyzes the
.beta.1-4 glycosidic bond between N-acetylglucosamine and
N-acetylmuramic acid.
[0136] For purposes of the present disclosure, "endopeptidase"
refers to any peptidoglycan hydrolytic enzyme that hydrolyzes any
of the bonds within the peptide subunits crosslinking glycan
strands.
[0137] For purposes of the present disclosure, "PlyG" refers to a
Bacillus phage gamma endolysin amidase (Schuch, R. et al.; A
Bacteriolytic Agent that Detects and Kills Bacillus Anthracis.
Nature. 2002, 418, 884-889).
[0138] For purposes of the present disclosure, "PlyPH" refers to a
Bacillus phage endolysin lysozyme (Yoong, P. et al.; PlyPH, a
Bacteriolytic Enzyme with a Broad PH Range of Activity and Lytic
Action again Bacillus Anthracis. J. Bacteriol. 2006, 188,
2711-2714). A non-limiting example of PlyPH may include
BCZK2532.
[0139] For purposes of the present disclosure, "similarity" refers
to the degree of resemblance between two or more amino acid,
peptide, polypeptide, or protein sequences or two or more nucleic
acid, DNA, nucleotide, or polynucleotide sequences, as determined
by the match between strings of such sequences. As a non-limiting
example, a peptide fragment, or the amino acid sequence within a
region of a full length protein is similar to that of another as
further defined by the degree to which amino acid residues within
said regions are conserved.
[0140] For purposes of the present disclosure, "identity" refers to
the condition of one particular amino acid, a peptide fragment, or
the amino acid sequence within a region of a full length protein
being the same to that of another amino acid, a peptide fragment,
or the amino acid sequence within a region of a full length
protein, as determined by the match between strings of such
sequences, after aligning the sequences and inserting gaps between
the sequence residues.
[0141] Identity and similarity may be determined by a variety of
methods including, but not limited to, those described in Dolz, R.;
GCG: Comparison of Sequences. In Methods in Molecular Biology
Computer Analysis of Sequence Data, Part I, Vol. 24; Humana Press,
Totowa, 1994; pp. 65-82; Dolz, R; GCG: Production of Multiple
Sequence Alignment. In Methods in Molecular Biology Computer
Analysis of Sequence Data, Part I, Vol 24; Humana Press, Totowa,
1994; pp. 83-99; and Carrillo, H.; Lipman, D.; The Multiple
Sequence Alignment Problem in Biology. J. App. Math. 1988, 48,
1073-1082.
[0142] Methods to determine identity may generally be designed to
give the largest match between the sequences tested. Methods to
determine identity and similarity may be codified in a variety of
computer programs including, but not limited to, GCG program
package (Devereux, J. et al.; A Comprehensive Set of Sequence
Analysis Programs for the VAX. Nucleic Acids Research. 1984, 12.1,
387-395), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et
al.; Basic Local Alignment Search Tool. J. Mol. Biol. 1990, 215,
403-410). The BLAST X program is publicly available from NCBI and
other sources (Altschul, S. F. et al.; Basic Local Alignment Search
Tool. J. Mol. Biol. 1990, 215, 403-410). The Smith Waterman
algorithm may optionally also be used to determine identity.
[0143] Parameters for polypeptide sequence comparison may include
those described in the following: Needleman, S. B.; Wunsch, C. D.;
A General Method Applicable to the Search for Similarities in the
Amino Acid Sequence of Two Proteins. J. Mol. Biol. 1970, 48,
443-453; Henikoff, S.; Henikoff, J. G.; Amino Acid substitution
Matrices from Protein Blocks. Proc. Natl. Acad. Sci. USA. 1992, 89,
10915-10919. As a non-limiting example, a program useful with these
parameters is the publicly available "Ogap" program from Genetics
Computer Group, located in Madison, Wis.
[0144] Parameters for nucleic acid comparison may include those
described in the following: Pearson, W. R.; Lipman, D. J.; Improved
Tools for Biological Sequence Comparison. Proc. Natl. Acad. Sci.
USA. 1988, 85, 2444-2448.
[0145] Optionally, in determining the degree of amino acid
similarity, conservative amino acid substitutions may also be
considered. Conservative amino acid substitutions generally refer
to the interchangeability of residues having similar side chains
and usually without affecting function. For example, a group of
amino acids having aliphatic side chains may include glycine,
alanine, valine, leucine, and isoleucine; a group of amino acids
having aliphatic-hydroxyl side chains may include serine and
threonine; a group of amino acids having amide-containing side
chains may include asparagine and glutamine; a group of amino acids
having aromatic side chains may include phenylalanine, tyrosine,
and tryptophan; a group of amino acids having basic side chains may
include lysine, arginine, and histidine; and a group of amino acids
having sulphur-containing side chains may include cysteine and
methionine. Conservative amino acids substitution groups include,
but are not limited to, valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. Substitutional variants of the amino acid
sequence disclosed herein are those in which at least one residue
in the disclosed sequences has been removed and a different residue
inserted in its place. In embodiments, the amino acid change may be
conservative. Conservative substitutions for naturally occurring
amino acids include, but are not limited to: alanine to serine;
arginine to lysine; asparagine to glutamine or histidine; aspartate
to glutamate; cysteine to serine or alanine; glutamine to
asparagine; glutamate to aspartate; glycine to proline; histidine
to asparagine or glutamine; isoleucine to leucine or valine;
leucine to isoleucine or valine; lysine to arginine, glutamine or
glutamate; methionine to leucine or isoleucine; phenylalanine to
methionine, leucine or tyrosine; serine to threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine; and, valine to isoleucine or leucine.
[0146] For purposes of this disclosure, a nucleic acid sequence or
nucleotide sequence is a series of letters indicating the order of
nucleotides or nucleic acids within a DNA (using GACT) or RNA
molecule (using GACU). The DNA or RNA molecule may be single or
double stranded, and may be genomic, recombinant, messenger RNA
(mRNA) or complementary DNA (cDNA).
[0147] For purposes of this disclosure, an amino acid sequence or
peptide sequence is a series of letters indicating the order of
amino acids within a polypeptide or protein molecule.
[0148] For purposes of this disclosure, a nucleic acid construct is
a nucleic acid molecule that may be isolated from a naturally
occurring gene or which may be modified to contain segments of
nucleic acids which are combined or juxtaposed in a manner which
would not otherwise exist in nature. Optionally, a nucleotide
sequence present in a nucleic acid construct may be operably linked
to one or more control sequences, which direct the transcription or
expression of messenger RNA that the cell machinery (ribosome) then
translates into a peptide or polypeptide in a cell.
[0149] For purposes of this disclosure, a control sequence may
include all DNA sequences which are necessary, for the expression
of a protein. At a minimum, the control sequences may include a
promoter and transcriptional and translational start and stop
signals. Optionally, a promoter represented by a nucleotide
sequence present in a nucleic acid construct may be operably linked
to another nucleotide sequence encoding a peptide or
polypeptide.
[0150] For purposes of this disclosure, a nucleic acid sequence is
operably linked when a control sequence is appropriately placed at
a position relative to the nucleotide sequence coding for a
polypeptide so the control sequence directs the
transcription/production/expression of the peptide or polypeptide
of the invention in a cell. Operably linked may also optionally
define a configuration in which a nucleic acid sequence is
appropriately placed at a position relative to another nucleic acid
sequence that codes for a functional domain such that a chimeric
polypeptide may be encoded in a cell.
[0151] For purposes of this disclosure, the expression of a protein
may include any step involved in the production of the peptide or
polypeptide including, but not limited to, transcription,
post-transcriptional modification, translation, post-translational
modification and secretion.
[0152] For purposes of this disclosure, a transformation refers to
a permanent or transient genetic change induced in a cell following
the incorporation of new DNA (i.e. DNA exogenous to the cell). When
the cell is a bacterial cell, the term usually refers to an
extra-chromosomal, self-replicating vector such as a plasmid which
harbors a selectable antibiotic resistance.
[0153] For purposes of this disclosure, an expression vector may be
any molecular construct which may be conveniently subjected to
recombinant DNA procedures and can bring about the expression of a
nucleotide sequence encoding a polypeptide of the invention in a
cell. A promoter refers to a nucleic acid fragment that functions
to control the transcription of one or more genes or nucleic acids,
located upstream with respect to the direction of transcription of
the transcription initiation site of the gene. The promoter region
is related to the binding site identified by the presence of a
sequence that is recognized as a binding site by DNA-dependent RNA
polymerase, transcription initiation sites, and any other DNA
sequences, including, but not limited to, transcription factor
binding sites, repressor and activator protein binding sites, and
any other sequences of nucleotides that acts directly or indirectly
to regulate the amount of transcription from the promoter. Within
the context of the invention, a promoter preferably ends at
nucleotide -1 of the transcription start site (TSS).
[0154] For purposes of this disclosure, a polypeptide is any
peptide, oligopeptide, polypeptide, amino acid gene product,
expression product, or protein. A polypeptide may be a naturally
occurring or synthetic molecule.
[0155] For purposes of this disclosure, a fusion protein is an
expression product resulting from the fusion of two or more nucleic
acid constructs, and may include, but is not limited to a fusion of
two or more polypeptides, fragment, variant, or derivative thereof
corresponding to the two or more nucleic acid constructs.
[0156] In one or more embodiments, a candidate lysin is identified
by comparing DNA or protein sequences of a bacteria with sequences
of a model lytic enzyme capable of targeting moieties in the cell
wall of a bacteria and selecting sequences having an identity
greater than about 50% or similarity greater than about 60% with
the sequences of the entire model lytic enzyme or selected portions
thereof which may be associated with the lytic activity of the
enzyme.
[0157] Non-limiting examples of model lytic enzymes include small
phage-produced proteins responsible for lysis of an infected host
cell, such as those described in Borysowski, J. et al.;
Bacteriophage Endolysins as a Novel Class of Antibacterial Agents.
2006. Exp. Biol. Med. 231, 366-377. In one or more embodiments,
model lytic enzymes optionally include one or more endolysins. An
endolysin is a lytic enzyme or protein typically produced by a
bacteriophage that degrades the infected bacterial cell wall,
facilitating release of new bacteriophage from the infected
bacterial cells at the end of the phage infection cycle. Usually,
fully functional endolysins accumulate in the infected bacterial
cytosol near the end of the phage infection cycle.
[0158] Comparisons can be performed using various approaches that
allow computationally identifying candidate lysins as described
herein.
[0159] For example, a comparison is performed by in silico computer
searches of a bacteria's genome using the sequences of the model
lytic enzyme as a parameter. Search tools include, but are not
limited to, the Basic Local Alignment Search Tool (BLAST) from the
National Institutes of Health. Several strategies can be employed
for the computer searches. In one or more embodiments, the
comparison by computer searches comprises performing an analysis of
genome annotations to identify candidate genes encoding lytic
proteins. Annotations also may optionally identify genes that
function in bacterial cell wall synthesis and processing and may
include lytic enzymes.
[0160] Another in silico approach for comparing can be performed by
directly comparing DNA sequences of model lytic enzymes or by
directly comparing the predicted amino acid sequences of such
proteins to predicted sequences in targeted genomes.
[0161] A further exemplary in silico approach for comparing can be
performed by predicting the protein structure of known lytic
proteins based on their predicted or determined amino acid
sequences, and then searching available sequence databases (e.g.
DNA databases) for sequences that encode proteins of similar
predicted structure. Successfully targeted genes will encode lysins
that exhibit lytic activity against a targeted bacterial
pathogen.
[0162] The comparisons can be performed by analysis based on local
protein or DNA alignment studies that compare relatedness (identity
and similarity) using a known sequence (e.g. model lytic protein
and/or a portion thereof), or compilation of sequences (e.g. one or
more model lytic protein and/or a portion thereof), used to search
any available database (e.g. using BLAST) that contains genetic
(DNA) or protein sequences or information to identify novel protein
sequences.
[0163] A comparison can be performed between nucleotide sequences
or between amino acid sequences of the model lytic enzyme and
selected bacterial genomes. The comparison may be done by
considering sequences covering the model lytic enzyme (e.g., the
entire model lytic enzyme) or the one or more portions of the model
lytic enzymes associated with the lytic activity of the model lytic
enzyme. Those portions can include not only the lytic domain of the
model lytic enzyme, but also additional domains responsible for
protein stability, three-dimensional structure, and any other
features associated with an active protein. Those portions can vary
from one model enzyme to another. In embodiments, selected genome
sequences from a bacteria at issue show at least 50% identity or
60% similarity or about a 50% similarity or about 60% identity in
outcome. In other aspects, sequences with 70% to 90% identity or
similarity or about 70% to 90% identity are selected. In further
aspects, a sequence having at least 90% identity or similarity and
in particular about 95% or higher identity or similarity are
selected.
[0164] For example, in an embodiment, basic alignment and local
basic alignments in programs such as Geneious 5.0.3 or ClustalW are
used to look for specific similarities. Using default settings,
proteins with pairwise identity of 50% or higher can be typically
selected.
[0165] For example, in embodiments wherein BLAST is used, proteins
with a "Total Score" of about 100 or more and/or an E value of
0.001 or lower are selected as candidate lysins.
[0166] In another aspect, identification includes performing a
plurality of comparisons using the same, substantially the same, or
different approaches. In some of those aspects, performing a
plurality of comparisons using different approaches may identify
candidate lysins that show less than 50% identity or 60% similarity
to a model lytic enzyme or selected portions thereof that may be
associated with lytic activity of the enzyme. In one or more
embodiments, a search can be limited to direct DNA sequence
comparisons that can limit the number of genes encoding these lytic
proteins.
[0167] In other aspects of the invention, identifying candidate
lysins is performed by doing a plurality of screen levels of the
genome sequences of the lysins of interest. As a non-limiting
example, a first screening of one or more genomes involves analysis
of provisionally identified annotated genes encoding any of the
four classes of lytic proteins, which optionally may include model
lytic enzymes. Bacteria from which the identified genes may be
obtained may include, but are not limited to bacterial,
bacteriophage, prokaryotic, and eukaryotic. The second screen may
optionally include a BLAST search of the one or more identified
genes against gene sequences encoding these lytic proteins in
bacteria that are closely related to the bacteria of interest. The
third screen may optionally include a search for genes that have
been implicated in cell wall biosynthesis or metabolism. The fourth
screen may optionally include comparing the amino acid sequences
encoded by the identified genes to the amino acid sequences of
other lytic proteins. The fifth screen may optionally include a
computational analysis of the amino acid sequences of other lytic
proteins to identify specific structural characteristics of this
class of proteins followed by a computational search of genomes to
identify genes encoding proteins that may have similar structure.
Bacteria from which the identified genes may be obtained may
include, but are not limited to bacterial, bacteriophage,
prokaryotic, and eukaryotic. As a non-limiting example, a small
number of endolytic proteins have been structurally characterized,
and the structures of portions of the proteins responsible for
lytic activity have been identified, from which a search for
similar structures encoded in the genomes of organisms of interest
can be performed. Exemplary procedures for identifying candidate
lysins are reported in the Examples of the present application.
[0168] Once a candidate lysin is identified, the candidate lysin
can be tested for lytic activity on the same bacteria expressing
the candidate lysin or on a bacteria related to the bacteria
expressing the candidate lysin. In one or more embodiments, the
gene coding for the candidate lysin can be amplified from a
bacterial genome, cloned and expressed in an in vitro transcription
(IVT) system. Exemplary procedures are illustrated in the Examples
of the present application.
[0169] In another embodiment, the identified candidate lysin is
acquired from other sources such as transformed host cells.
[0170] In one or more embodiments, the candidate lysin is subjected
to one or more tests, where the bacteria is exposed to the
candidate lysin to determine the lytic activity of the candidate
lysin on the bacteria. In a further embodiment, a first screen is
provided by a spot test. Although this procedure can be performed
with a substantially pure protein, or a purified protein, the
procedure does not require purified protein. Additional procedures
that may be used to detect lytic activity and to determine
suitability as lysins include, but are not limited to, the lysis of
the bacteria, a visible clearing or reduction of viable cells, or a
decrease in the absorbance of a cellular suspension exposed to the
lysin as recorded with a spectrophotometer.
[0171] Once one or more candidate genes are identified at each
step, the genes can be amplified from the bacterial genome of
interest in a single set of experiments. In one or more
embodiments, PCR primers suitable to amplify the genes of interest
and facilitate their (immediate) cloning into a standard expression
vector are used to stream-line the process. Additionally, several
gene products can be tested simultaneously.
[0172] Additionally, where the amplification is performed from the
genome of a bacteria other than the original bacteria expressing
the enzyme, a factor that may be considered are codon preferences
of the target microbe from which the gene is amplified relative to
those where the amplification is performed. If codon preferences
are significantly different between the original bacteria and the
microbe from which the putative lytic protein gene is cloned and
expressed, that gene may not be expressed to a desired level in the
selected bacteria. Other approaches can be used in addition to or
as an alternative to include, but are not limited to, an IVT
system. These approaches include, but are not limited to,
re-synthesizing the gene using chemical DNA synthesis to produce a
gene that encodes the same protein amino acid sequence but that is
optimized for expression in the recombinant organism system.
[0173] The candidate lysins that have a detectable lytic activity
can be selected as lysins for one or more species of bacteria
wherein the lytic activity has been tested or can be predicted
based on genetic relationship.
[0174] The lysins obtained by one or more of the methods described
comprise proteins that possess enzymatic and lytic activity that
specifically damage cell walls and that destroy the bacterial
target. Non-limiting examples of the lysins that may be obtained by
methods disclosed are structurally related to lysozymes (also known
as muramidase) or other model lytic proteins. These model lytic
proteins are comprised of an N-terminal portion (domain)
responsible for lytic activity and a C-terminal portion (domain)
responsible for target recognition and binding. In various
embodiments, the lysins obtained by these one or more of the
methods described differ from lysozymes or other model lytic
proteins because of their high specificity and ability to target a
given bacteria or group of bacteria based on the chemical structure
and linkage of cell wall components.
[0175] The lysins described in this disclosure differ from other
lysins including, but not limited to model lytic proteins in that,
under certain conditions, they are more efficacious than the model
lytic proteins and are encoded by genes from the target bacteria's
own genome. The lysins described herein may be comprised of lytic
proteins which target specific moieties of the bacteria's cell wall
and normally play a role in synthesizing and maintaining the
peptidoglycan layer of a bacteria's cell wall. In general the
lysins described in this disclosure are enzymes encoded by the
target bacteria's own genes and are involved in cell wall
biosynthesis and metabolism.
[0176] The lysins described in this disclosure are able to disrupt
and destabilize the peptidoglycan cell wall of the bacteria
expressing the lysin under suboptimal conditions for a bacteria to
regulate lysin expression and/or activity, which may result in
preventing the bacteria from maintaining osmotic equilibrium or
which may otherwise impact the viability of the cell.
[0177] The suboptimal conditions for a bacteria to regulate lysin
expression and/or activity may include, but are not limited to
conditions where the concentrations of lysin obtained by one or
more of the methods described is higher than the concentration of
lysin produced by the targeted bacteria, which are not confined to
the locations within the cells of the bacteria where they are
normally expressed, absence of inhibitors, and other conditions. In
one or more embodiments where the bacteria is a spore-forming
bacteria, the conditions typically include any condition where the
spore-forming bacteria are in a vegetative state. In other
embodiments, non-appropriate conditions for a bacteria to regulate
lysin expression and/or activity may include, but are not limited
to providing a concentration of a lysin that is about 2:1 or
greater than the concentration produced by the bacteria. In several
embodiments, exposure of the bacteria to concentrations of a lytic
protein described in this disclosure that are at least 2-3 orders
of magnitude higher than the concentration normally produced by the
bacteria or a bacteria that is genetically related to the bacteria
expressing a lytic protein described in this disclosure, typically
results in rapid cell lysis and death of the bacteria or the
bacteria that is genetically related. A non-limiting example of
concentrations of lysin normally produced by the bacteria includes
production of very minute amounts at very specific locations within
the bacterial cells.
[0178] The lysins described in this disclosure may kill or inhibit
growth not only of the bacteria expressing the enzyme but also of
genetically related bacteria.
[0179] Generally, genetically related bacteria express at least one
protein that has a 50% or higher identity or a 60% or higher
similarity or about a 50% or higher identity or about a 60% or
higher similarity to a corresponding protein in another bacteria.
As a non-limiting example, a first bacteria expressing a first
lysin is related to a second bacteria expressing a second lysin, if
the second protein has about a 50% or higher identity or a 60% or
higher similarity to the first lysin. A closer genetic relationship
is evidenced in some embodiments, by sequences showing a 70% to 90%
identity or similarity or about 70% to 90% identity or similarity,
sequences having at least 90% identity or similarity and in
particular about 95% or higher identity or similarity between the
bacteria.
[0180] For example, members of a subgroup of Bacillus, which
includes Bacillus anthracis, are genetically related with a subset
of Bacillus species. As a non-limiting example, Bacillus cereus is
closely related but Bacillus subtilis is not as closely related to
Bacillus anthracis. Similarly, Yersinia pestis is very closely
related to Yersinia pseudotuberculosis but not as closely related
to Yersinia enterocolitica. Burkholderia mallei and Burkholderia
pseudomallei are very closely related while neither is closely
related to Bacillus anthracis.
[0181] The effectiveness of a lysin on a bacteria related to
another bacteria expressing the lysin is correlated to how closely
related the bacteria are. Accordingly, the higher the percentage
identity/similarities between at least one lysin protein expressed
by both of the bacteria, the higher the effectiveness of the lysin
is expected.
[0182] Lytic and endolysin activity are forms of hydrolytic
activity that damages or destroys specific linkages between cell
wall moieties resulting in degradation of the cell wall and
resulting bacterial cell death. Endolysins are produced by a
bacteriophage during the bacterial lytic infection cycle and serve
to disrupt the bacterial cell wall to release progeny phage. Both
endolysin activity and lytic activity modify, alter, or damage
bacterial cell walls. Lytic proteins may be derived from non-phage
sources. These proteins normally function, in vivo, to regulate
cell wall synthesis and maintenance, but may also be used to
generate unregulated breaks in the cell wall, usually resulting in
disruption of growth and also cell death. Typically, lytic and
endolysin activity is associated with a protein that is a member of
one of four classes of peptidoglycan hydrolases and possibly, some
as yet uncharacterized proteins.
[0183] Differences in activity between lytic enzymes may be
measured using various technical approaches. As a non-limiting
example, the difference in activity between a model lytic enzyme
and a lysin or between two lysins described in this disclosure may
be measured by applying the same concentration of lysin to the same
number of bacterial cells in the same reaction volume and
incubating under the optimal conditions for each lysin for a
specific period of time. The number of surviving cells is then
measured by plating the treated cells on agar plates and counting
the number of colonies that grow on the plates. If identical
concentrations of the two proteins result in a difference in the
number of surviving colonies the relative effectiveness of the
lysins may be determined. As a non-limiting example, if treatment
with a phage endolysin results in 100 colonies growing on the test
plate and 0 colonies growing on the bacterial lysin test plate,
then the bacterial lytic protein is at least 100 times or two
orders of magnitude (10.times.10) more effective than the phage
endolysin infection cycle.
[0184] In one or more embodiments, the lysins described in this
disclosure are recombinant proteins, which can be identical or
modified with respect to the original enzyme. As a non-limiting
example, a recombinant lysin may comprise a six histidine residue
end tag to facilitate rapid purification of the protein. Other
non-limiting examples include those modifications to improve
solubility or other features of the lysins. As a non-limiting
example, in some embodiments lytic proteins are not soluble at a
desired concentration including, but not limited to low nM or pM
concentrations. In a non-limiting example, modifications of the
gene encoding the protein, including, but not limited to the
addition of other genetic sequences that facilitate protein
solubility can be used to enhance the solubility properties of the
lytic proteins.
[0185] In an alternative embodiment, the lytic proteins described
in the disclosure may optionally be modified to adapt the
DNA/protein sequence to the non-native system so that the protein
may be produced in a non-native system. As a non-limiting example,
the DNA sequence of the gene encoding a lytic protein that destroys
methicillin-resistant S. aureus (MRSA) cells is not efficiently
expressed in an Escherichia coli-based IVT system because E. coli
has a preference for specific nucleotide triplets when producing
proteins that are different from those preferred by S. aureus. By
modifying the DNA sequence of the gene encoding a lytic protein for
optimal expression in the E. coli IVT system, production of the
lytic protein that is identical to that encoded by the S. aureus
gene may be enhanced. Further, in an alternate embodiment, the
lysin may be modified by manipulating the DNA sequence of the
portion of the gene that encodes the C-terminal end of the lytic
protein because this part of the protein provides target
specificity to the enzyme, thereby producing a chimeric protein
that attacks specific moieties of a bacterial cell wall in a
different species of bacteria.
[0186] In an alternative embodiment, the lytic proteins described
in the disclosure may include, but are not limited to one or more
fragments comprising consecutive amino acid residues from at least
7 or more to less than all, at least 10 or more to less than all,
at least 15 or more to less than all, preferably at least 20 or
more to less than all, at least 25 or more to less than all, more
preferably at least 35 or more to less than all, at least 40 or
more to less than all, or at least 50 or more to less than all of
amino acids constituting the lytic protein, or a variant thereof,
and retains the one or more functional domains, or does not affect
the functionality of the lytic protein.
[0187] In an alternative embodiment, the lytic proteins described
in the disclosure may include, but are not limited to one or more
variants comprising an amino acid sequence derived from the amino
acid sequence of the lytic protein, or a fragment thereof, by the
deletion, substitution, addition, or insertion of one or more amino
acids, or to one or more variants that exhibit at least 50%
identity or 60% similarity, preferably at least 70% to 90% identity
or similarity, more preferably at least 90% identity or similarity,
and more preferably at least 95% identity or similarity to the
amino acid sequence of the lytic protein, or a fragment
thereof.
[0188] Bacteria that can be targeted by the lysins of the invention
may include numerous prokaryotic microbial species. The amide bond
(CO--NH) targeted by amidases is located between N-acetylmuramic
acid in the glycan chain and L-alanine in the cross-linking peptide
subunit. The amide bond may be present in the peptidoglycan
structure of both Gram-positive and Gram-negative bacteria from
numerous species within various phyla, including, but not limited
to Acidobacteria, Actinobacteria, Bacteroidetes, Chlamydiae,
Cyanobacteria, Deinococcus-Thermus, Firmicutes, Fusobacteria,
Nitrospira, Planctomycetes, Proteobacteria, and Spirochaetes. The
phylum Proteobacteria includes, but is not limited to purple
photosynthetic and non-photosynthetic gram-negative bacteria,
including cocci, non-enteric rods and enteric rods, such as, for
example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia,
Francisella, Haemophilus, Bordetella, Escherichia, Salmonella,
Shigella, Klebsiella, Proteus, Pseudomonas, Bacteroides,
Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla,
Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema and
Fusobacterium.
[0189] An alternative embodiment further relates to an isolated
nucleic acid molecule encoding the polypeptide, fragment, variant,
or derivative thereof or a fusion of the polypeptide, fragment,
variant, or derivative. Non-limiting examples of isolated and/or
purified nucleic acid molecules according to the present disclosure
include a nucleic acid sequence according to SEQ ID NO: 1. One or
more embodiments of the disclosure further relates to a recombinant
vector comprising the nucleic acid molecule. The vector may provide
for the constitutive or inducible expression of one or more
polypeptides, fragments, variants, or derivatives thereof or a
fusion of the polypeptide, fragment, variant, or derivative. One or
more embodiments of the disclosure also relates to a method for
obtaining one or more polypeptides, fragments, variants derivatives
or a fusion of the polypeptide, fragment, variant, or derivative
from a microorganism, such as a genetically modified suitable host
cell which expresses one or more polypeptides, fragments, variants,
derivatives or a fusion of the polypeptide, fragment, variant, or
derivative. The host cell may be a microorganism such as bacteria
or yeast or an animal cell as e.g., a mammalian cell, e.g., a human
cell. In one or more embodiments, the host cell is an E. coli cell.
The host may be selected for biotechnological reasons, including,
but not limited to yield, solubility, and costs and may also be
selected for medical reasons, including, but not limited to a
non-pathological bacteria or yeast or human cells. One or more
embodiments of the disclosure further relates to a method for
genetically transforming a suitable host cell in order to obtain
the expression of the polypeptide, fragment, variant, or derivative
thereof or fusion of the polypeptide, fragment, variant, or
derivative, wherein the host cell is genetically modified by the
introduction of a genetic material encoding one or more
polypeptides, fragments, variants, or derivatives thereof or a
fusion of the polypeptide, fragment, variant, or derivative into
the host cell and obtain their translation and expression
products.
[0190] The lysins described in this disclosure can be used to
disrupt Bacillus anthracis cell walls, thus avoiding the use of the
conventional methods for lysis of Bacillus anthracis cells, which
do not provide consistent results essential for comparing genome
copy numbers from cells grown with and without antibiotic. As a
non-limiting example, conventional methods include, but are not
limited to boiling, bead heating, and sonication.
[0191] In one or more embodiments, identification of a novel lysin
protein may involve the review of whole genome databases for
Bacillus anthracis and near neighbors; the selection of genes
encoding potential lytic enzymes based on conserved amino acid
motifs found in peptidoglycan hydrolases; the cloning of selected
genes in an expression vector, the isolation of the proteins, and
the testing for lytic activity against Bacillus anthracis.
Candidates genes with highest activity as optionally determined by
visible clearing or reduction of viable cells or by a decrease in
the absorbance of a cellular suspension exposed to the lysin as
recorded with a spectrophotometer, are selected, scaled up for
production and purification, and optimized for lysis conditions. In
one or embodiments, the preceding order is followed.
[0192] A novel lytic enzyme designated BQ22 was selected from five
identified candidate genes: four from Bacillus anthracis and one
from Bacillus weihenstephanensis. After cloning, enzymatic activity
evaluation, protein solubility assessment, buffer optimization,
lyophilization conditions of the candidate genes and gene products
was performed, and stability studies were conducted.
[0193] The BQ22 lysin protein gene construct may comprise SEQ ID
NO: 1 which optionally includes, but is not limited to a T7
promoter region (SEQ ID NO: 7), a His-Tag sequence (SEQ ID NO: 5),
a lac operator (SEQ ID NO: 6), and a stop codon (see, for example,
FIG. 9).
[0194] In one or more embodiments, the disclosure provides a newly
characterized lytic enzyme BQ22 lysin. The BQ22 lysin and several
engineered derivatives were expressed in E. coli and showed
surprising stability after lyophilization as proven by their lytic
activity after reconstitution. The new BQ22 lysin has excellent
lytic activity against Bacillus anthracis.
[0195] In one or more embodiments, the new BQ22 lysin is an
important component in a rapid antimicrobial susceptibility test
developed for Bacillus anthracis. As shown in Table 1 below,
conventional susceptibility testing of this species requires 16-20
hours of incubation time because the testing relies on visible
growth for interpretation of the results. In one or more
embodiments, conventional susceptibility testing includes, but is
not limited to, broth microdilution, Etest, agar dilution, or disk
diffusion. The novel lysins described in this disclosure,
including, but not limited to the BQ22 lysin provides a
mechanism/reagent necessary to ensure the consistent, reproducible
cell lysis that is required to perform the rapid test in only 6
hours that uses real-time PCR to detect growth after an abbreviated
incubation time.
TABLE-US-00001 TABLE 1 Time Required for Susceptibility Testing
Incubation Incubation for Incubation for rapid for conventional
susceptibility Agent* Isolation * susceptibility testing testing B.
anthracis 16 to 20 h 16 to 20 h 4 h Y. pestis 48 h 24 to 48 h 6 h
F. tularensis 48 to 72 h 48 h 12 h B. pseudomallei 16 to 20 h 16 to
20 h 6 h B. mallei 16 to 20 h 16 to 20 h 6 h * A pure culture is
required for susceptibility testing
[0196] The present disclosure provides various embodiments as
described below. However, it should be noted that the present
disclosure is not limited to the embodiments described herein.
[0197] A Bacillus lysin protein has been engineered and employed as
a component of a rapid antimicrobial susceptibility assay, as shown
in FIG. 1. FIG. 1 is a diagram showing a rapid antimicrobial
susceptibility assay 100 according to one or more embodiments. In
FIG. 1, the process begins at 105, by preparing a cell suspension
with a concentration of approximately 5.times.10.sup.5 cfu/ml. The
suspension is then aliquoted into susceptibility testing plates at
110, a process that takes about 15 minutes. In one or more
alternative embodiments, the cell suspension may comprise Bacillus
anthracis. The inoculum is added to the plates which contain
two-fold doubling dilutions of various antibiotics in the medium.
These plates are then incubated at 115, at 35.degree. C., for
approximately 4 hours. The cells in the inoculated plate are then
processed at 120 with BQ22 lysin for 15 minutes to release the DNA.
Then, at 125, real-time PCR reactions are set up using the cell
lysate produced by activity of BQ22, and the reaction mixtures are
subjected to 45 cycles of amplification, which requires about 1.5
hours, after which the data are analyzed.
[0198] FIG. 2 is a flow diagram showing a method of selecting a
lysin agent for use in molecular diagnostic testing according to
one or more embodiments of the present invention. As shown, the
method 200 includes operation 205 where genome databases for
Bacillus anthracis and near neighbors are analyzed. Next, the
process continues to operation 210 where candidate genes encoding
potential lytic enzymes are selected based on conserved amino acid
motifs that are characteristic of peptidoglycan hydrolases.
[0199] From operation 210 the process continues to operation 215
where the candidate genes are cloned in an expression vector and
proteins thereof are isolated and tested for lytic activity against
Bacillus anthracis.
[0200] Next from operation 215, the process continues to operation
220 where an optimum gene of the candidate genes is selected for
optimizing lysis conditions.
[0201] Approximately 140 novel lysin candidates were identified. Of
the 140 candidates, 13 candidates (Tables 2 and 3 shown below) were
cloned, sequenced, and tested for activity against Bacillus
anthracis. A 96-well plate format assay was created to
simultaneously test the activity of multiple lysin proteins against
Bacillus anthracis under multiple conditions.
TABLE-US-00002 TABLE 2 Potential Candidate Lysin Proteins Examined
In Vitro. (No Activity Observed) Protein Organism Accession No.
Predicted Function Testing Conditions BAH_A0050 Bacillus anthracis
ZP_02395105 Endopeptidase, 50 mM phosphate, (pH 5.0, pH 6.0, pH
7.0, str. A0442 Amidase pH 8.0) @ 35.degree. C., +/-0.1% Triton
X-100; phosphate-buffered saline, (pH 7.4) @ 35.degree. C., +/-0.1%
Triton X-100; 50 mM Tris buffer, pH 6.5, pH 7.5, pH 8.5 @
35.degree. C., +/-0.1% Triton X-100; 50 mM Tris, 100 mM NaC l @
35.degree. C., +/-0.1% Triton X-100 pE33L466_0234 Bacillus cereus
E33L YP_245729.1 Conserved 50 mM phosphate, (pH 5.0, pH 6.0, pH
7.0, hypothetical pH 8.0) @ 35.degree. C., +/-0.1% Triton X-100;
protein phosphate-buffered saline, (pH 7.4) @ 35.degree. C.,
+/-0.1% Triton X-100; 50 mM Tris buffer, pH 6.5, pH 7.5, pH 8.5 @
35.degree. C., +/-0.1% Triton X-100; 50 mM Tris, 100 mM NaCl @
35.degree. C., +/-0.1% Triton X-100 BA_0796 Bacillus anthracis str
NP_843315. 1 Hypothetical 50 mM phosphate, (pH 5.0, pH 6.0, pH 7.0,
Ames protein pH 8.0) @ 35.degree. C., +/-0.1% Triton X-100;
phosphate-buffered saline, (pH 7.4) @ 35.degree. C., +/-0.1% Triton
X-100; 50 mM Tris buffer, pH 6.5, pH 7.5, pH 8.5 @ 35.degree. C.,
+/-0.1% Triton X-100; 50 mM Tris, 100 mM NaCl @ 35.degree. C.,
+/-0.1% Triton X-100 BA_5104 Bacillus anthracis NP_847290. 1
D-Ala-D-Ala 50 mM phosphate, (pH 5.0, pH 6.0, pH 7.0, str. Ames
carboxypeptidase pH 8.0) @ 35.degree. C., +/-0.1% Triton X-100;
phosphate-buffered saline, (pH 7.4) @ 35.degree. C., +/-0.1% Triton
X-100; 50 mM Tris buffer, pH 6.5, pH 7.5, pH 8.5 @ 35.degree. C.,
+/-0.1% Triton X-100; 50 mM Tris, 100 mM NaCl @ 35.degree. C.,
+/-0.1% Triton X-100 BceRKBAB4_2909 Bacillus ABY44097.1 amidase 50
mM phosphate, (pH 5.0, pH 6.0, pH 7.0, weihenstephanensis pH 8.0) @
35.degree. C., +/-0.1% Triton X-100; KBAB4 phosphate-buffered
saline, (pH 7.4) @ 35.degree. C., +/-0.1% Triton X-100; 50 mM Tris
buffer, pH 6.5, pH 7.5, pH 8.5 @ 35.degree. C., +/-0.1% Triton
X-100; 50 mM Tris, 100 mM NaCl @ 35.degree. C., +/-0.1% Triton
X-100 BA_0224 Bacillus anthracis AAP24267 lysozyme-like 50 mM
phosphate (pH 6.0, pH 7.0, pH 8.0,) +/-0.1% str. Ames LYZ domain
Triton X-100, +/1 1 mM DTT, @ 28.degree. C.; phosphate-buffered
saline (PBS) pH 7.4, +/-0.1% Triton-X, +/-100 mM DTT, @ 28.degree.
C.; 50 mM Tris (pH 6.5, pH 7.5, pH 8.5), +/-1 mM DTT, @ 28.degree.
C.; 50 mM Tris (pH 7.5, pH 8.5), +/-0.1% Triton X-100, +/-1 mM
metals @ 28.degree. C.; 50 mM Tris (pH 7.5, pH 8.5) +/-100 mM NaCl,
+/-1 mM DTT, +/-0.1% Triton X-100; 50 mM MES pH 5.2, +/-0.1% Triton
X-100, +/-1 mM DTT, +/-1 mM metals @ 28.degree. C.; 50 mM sodium
acetate pH 5.2, +/-0.1% Triton X-100, +/-1 mM DTT, +/-1 mM metals,
@ 28.degree. C. BA_3893 Bacillus anthracis NP_846140 Putative cell
wall 50 mM phosphate (pH 6.0, pH 7.0, pH 8.0,) +/-0.1% str. Ames
hydrolase Triton X-100, +/1 1 mM DTT, @ 28.degree. C.;
phosphate-buffered saline (PBS) pH 7.4, +/-0.1% Triton-X, +/-100 mM
DTT, @ 28.degree. C.; 50 mM Tris (pH 6.5, pH 7.5, pH 8.5), +/-1 mM
DTT, @ 28.degree. C.; 50 mM Tris (pH 7.5, pH 8.5), +/-0.1% Triton
X-100, +/-1 mM metals @ 28.degree. C.; 50 mM Tris (pH 7.5, pH 8.5)
+/-100 mM NaCl, +/-1 mM DTT, +/-0.1% Triton X-100; 50 mM MES pH
5.2, +/-0.1% Triton X-100, +/-1 mM DTT, +/-1 mM metals @ 28.degree.
C.; 50 mM sodium acetate pH 5.2, +/-0.1% Triton X-100, +/-1 mM DTT,
+/-1 mM metals, @ 28.degree. C. BA_3698 Bacillus anthracis
NP_845961 amidase 50 mM phosphate (pH 6.0, pH 7.0, pH 8.0,) +/-0.1%
str. Ames Triton X-100, +/1 1 mM DTT, @ 28.degree. C.;
phosphate-buffered saline (PBS) pH 7.4, +/-0.1% Triton-X, +/-100 mM
DTT, @ 28.degree. C.; 50 mM Tris (pH 6.5, pH 7.5, pH 8.5), +/-1 mM
DTT, @ 28.degree. C.; 50 mM Tris (pH 7.5, pH 8.5), +/-0.1% Triton
X-100, +/-1 mM metals @ 28.degree. C.; 50 mM Tris (pH 7.5, pH 8.5)
+/-100 mM NaCl, +/-1 mM DTT, +/-0.1% Triton X-100; 50 mM MES pH
5.2, +/-0.1% Triton X-100, +/-1 mM DTT, +/-1 mM metals @ 28.degree.
C.; 50 mM sodium acetate pH 5.2, +/-0.1% Triton X-100, +/-1 mM DTT,
+/-1 mM metals, @ 28.degree. C. BA_1818 Bacillus anthracis
NP_844239.1 glucosaminidase 50 mM phosphate (pH 6.0, pH 7.0, pH
8.0,) +/-0.1% str. Ames Triton X-100, +/1 1 mM DTT, @ 28.degree.
C.; phosphate-buffered saline (PBS) pH 7.4, +/-0.1% Triton-X,
+/-100 mM DTT, @ 28.degree. C.; 50 mM Tris (pH 6.5, pH 7.5, pH
8.5), +/-1 mM DTT, @ 28.degree. C.; 50 mM Tris (pH 7.5, pH 8.5),
+/-0.1% Triton X-100, +/-1 mM metals @ 28.degree. C.; 50 mM Tris
(pH 7.5, pH 8.5) +/-100 mM NaCl, +/-1 mM DTT, +/-0.1% Triton X-100;
50 mM MES pH 5.2, +-0.1% Triton X-100, +/-1 mM DTT, +/-1 mM metals
@ 28.degree. C.; 50 mM sodium acetate pH 5.2, +/-0.1% Triton X-100,
+/-1 mM DTT, +/-1 mM metals, @ 28.degree. C.
[0202] Three of the 13 novel lysin candidates, BceRKBAB4_3364,
BA_0898, and BA_2528, (Table 3 shown below) were found to hydrolyze
Bacillus anthracis peptidoglycan, as demonstrated by a decrease in
absorbance at A600 nm, with BA_2528 having the greatest activity
when tested with crude lysates of E. coli expressing the lysin
protein. BA_2528 is a predicted amidase of molecular mass (MM)
.about.43 kDa (FIGS. 3-4), that is shown herein to be active
against vegetative cells of Bacillus anthracis Sterne (FIG. 5).
TABLE-US-00003 TABLE 3 Candidate Lysin Proteins Examined In Vitro
Having Activity Against Bacillus anthracis. Expressed Predicted
from Protein ID Organism Accession No. Function Activity Notes
Plasmid BceR Bacillus YP_001646166.1 muramidase Maximum activity at
50 mM pBQ11 KBAB4_3364 Weihenstephanensis phosphate, pH 5.0 @
35.degree. C. KBAB4 Conditions tested: 50 mM phosphate (pH 5.0, pH,
6.0, pH 7.0, pH 8.0) @ 30.degree. C. and @ 35.degree. C.; +/-0.1%
Triton X-100; 50 mM Tris (pH 6.5, pH 7.5, pH 8.5), +/-0.1% Triton
X-100, +/-100 mM NaC1, @ 30.degree. C. and 35.degree. C. BA_0898
Bacillus anthracis NP_843409 amidase Maximum activity at 50 mM
pBQ15 str. Ames Tris, pH 8.5 w/0.1% Triton X- 100. Conditions
tested: 50 mM phosphate (pH 5.0, pH, 6.0, pH 7.0, pH 8.0) @
30.degree. C. and @ 35.degree. C.;, +/-0.1% Triton X- 100; 50 mM
Tris (pH 6.5, pH 7.5, pH 8.5), +/-0.1% Triton X- 100, +/-100 mM
NaC1, @ 30.degree. C. and 35.degree. C. BA_2528* Bacillus anthracis
NP_844897 amidase Maximum activity at 50 mM pBQ13 str. Ames Tris,
pH 7.5-8.5. Conditions PBQ22 tested: 50 mM phosphate (pH pBQ23 5.0,
pH 6.0, pH 7.0, pH 8.0), +/-0.1% Triton X-100, @ 35.degree. C;
phosphate-buffered saline (PBS), pH 7.4, +/-0.1% Triton X-100, @
35.degree. C; 50 mM Tris (pH 6.5, pH 7.5, pH 8.5), +/-0.1% Triton
X-100, +/-100 mM NaCl, @ 35.degree. C; 50 mM Tris (pH 8.5), +/-1 mM
EDTA; 50 mM Tris (pH 7.5), 1% Triton X-100, 1 mM or 10 mM of one of
the following (ZnCl.sub.2, MnCl.sub.2, CaCl.sub.2, or
MgCl.sub.2).
[0203] In FIG. 3, the arrows denote induction products for BQ22
(BA_2528 N-terminal 6.times.His) in lanes 1 and 2 (shown as
pBQ22.3.1 and pBQ22.3.2 respectively), BQ23 (BA_2528 C-terminal
6.times.His) in lanes 3 and 4 (shown as pBQ23.3.1 and pBQ23.3.2
respectively), BQ24 (BA_2528 Catalytic domain only) in lanes 5 and
6 (shown as pBQ24.3.1 and pBQ24.3.2 respectively), BQ13 (BA_2528
wild type) in lane 8 (shown as pBQ13), and BQ16 (BCZK2532 wild
type) in lane 9 (shown as pBQ16) with Molecular Mass markers in
kiloDaltons (kDa) in lane 1 and pET16b vector control in lane 10 on
a SDS-PAGE gradient gel.
[0204] In FIG. 4, the arrows denote induction product for BQ22
(BA_2528 N-terminal 6.times.His) in lanes 1 and 2 (shown as
pBQ22.3.1 and pBQ22.3.2 respectively), BQ23 (BA_2528 C-terminal
6.times.His) in lanes 4 and 5 (shown as pBQ23.3.1 and pBQ23.3.2
respectively), BQ24 (BA_2528 Catalytic domain only) in lanes 6 and
7 (shown as pBQ24.3.1 and pBQ24.3.2 respectively), BQ13 (BA_2528
wild type) in lane 8 (shown as pBQ13), and BQ16 (BCZK2532 wild
type) in lane 9 (shown as pBQ16) with Molecular Mass markers in
kiloDaltons (kDa) in lane 3 and pET16b vector control in lane 10 on
a SDS-PAGE gradient gel.
[0205] FIG. 5 illustrates a graph showing lysin activity data from
testing crude lysates of IPTG-induced E. coli that were incubated
with vegetative cells of Bacillus anthracis Sterne at 35.degree. C.
over time at the buffer conditions indicted beside the graph. The
decrease in A600 was monitored with a Spectramax 384 Plus
Spectrophotometer. Samples tested were BQ13 (BA_2528 wild type)
shown as pBQ13, BQ22 (BA_2528 N-terminal 6.times.HisTag) shown as
pBQ22.3.1 and pBQ22.3.2, BQ23 (BA_2528 C-terminal 6.times.HisTag)
shown as pBQ23.3.1 and pBQ23.3.2, BQ24 (BA_2528 Catalytic domain
only, untagged) shown as pBQ24.3.1 and pBQ24.3.2, BQ16 (BCZK2532,
positive control) shown as pBQ16, and pET16b (vector control) shown
as pET16b. Addition of BCZK2532 caused all cells to lyse before
recording could begin, and thus the curve appears as a flat
line.
[0206] FIG. 6 illustrates a comparison of 1 or 2 .mu.g of
Immobilized Metal Ion Affinity Chromatography (IMAC)-purified
proteins on a 4-15% gradient SDS-PAGE gel, from the 6.times.HisTag
constructs BQ22 (lanes 2 and 6), BQ23 (lanes 3 and 7), BCZK2532
(lanes 4 and 8), 1 .mu.g from the pET16b vector control (lanes 5
and 9), respectively. Molecular mass markers are located in lane 1
on this gel. Lanes 2, 3, and 4 contained 1 .mu.g protein and lanes
6, 7, and 8 contain 2 .mu.g protein.
[0207] FIG. 7 illustrates enzyme activity for normalized molar
amounts of purified BQ22 (BA_2528 N-terminal 6XH) shown as BQ22_6H
R1, BQ23 (BA_2528 C-terminal 6XH) shown as BQ23_6H R1, and BCZK2532
shown as BCK2532_6H (positive control for purification and
activity), and pET16b (vector control) shown as pET16b, added to
exponential phase Bacillus anthracis Sterne in 50 mM Tris, pH 7.5,
containing 0.1% Triton X-100 at 35.degree. C. The decrease in
absorbance at A600 nm was monitored with a Spectramax 384 Plus
Spectrophotometer. R1 denotes the use of only one of the two
replicates when plotting data. The addition of BCZK2532 caused all
cells to lyse before recording could begin, and thus the curve
appears as a flat line.
[0208] The IMAC purified BQ22 is also active against Yersinia
pestis; see for example, FIG. 8, wherein 500 ng of IMAC-purified
BQ22 was added to EDTA-treated Yersinia pestis cells at the
conditions shown in Wash buffer (W, to remove EDTA) and Assay
buffer (A), which were mixtures of Tris, pH 7.5 or pH 8.0, with 1%
or 0.1% Triton X-100 with or without either 0.1% or 1.0% Triton
X-100. Wash buffer (W) and assay buffer (A) were Tris, pH 7.5 or pH
8.0. The results illustrate a decrease in absorbance at A600 nm, as
monitored by a Spectramax 384 Plus Spectrophotometer (Molecular
Devices, LLC, Sunnyvale, Calif.).
[0209] The BCZK2532 protein is encoded on the B. cereus E33L
genome, and the protein differs by only one conservative amino acid
change from the same protein encoded by gamma phage on the Bacillus
anthracis genome. B. cereus E33L is the closest known relative of
Bacillus anthracis. The closest known relatives of B. cereus E33L
are Bacillus anthracis and B. thuringiensis 97-27 as defined by an
abundance of phylogenetic and genome sequencing criteria. In
contrast, B. thuringiensis HD1 and HD560 are only distantly related
to B. cereus E33L while B. thuringiensis HD658 is more closely
related but not as closely related as the other two isolates.
Non-limiting experiments described in this disclosure, including,
but not limited to those related to percent survival as a function
of relatedness, indicate that the more closely related an isolate
is to the bacterial strain from which the lytic protein gene was
derived, the more effective the lytic protein is in lysing cells of
that isolate.
[0210] In a further aspect, the lysins can be used to target other
Gram-negative bacteria such as Yersinia pestis, Francisella
tularensis, Burkholderia pseudomallei and other Gram-negative
bacilli and cocci. Non-limiting examples include, but are not
limited to, Hemophilus influenzae, Klebsiella pneumoniae,
Legionella pneumophila, Pseudomonas aeruginosa, Escherichia coli,
Proteus mirabilis, Enterobacter cloacae, Serratia marcescens,
Helicobacter pylori, Salmonella enteritidis, and Salmonella typhi.
Polymyxins and other antibiotics that disrupt the structure of
Gram-negative outer membranes can be used against Gram-negative
bacteria, however, these antibiotics are relatively neurotoxic and
nephrotoxic, so high concentrations may be contraindicated in some
circumstances. In one or more embodiments, very low concentrations
of these antibiotics in combination with the lytic proteins
described in this disclosure, including, but not limited to BQ22
lysin are expected to result in rapid destruction of the
Gram-negative pathogen. Low concentrations of polymyxins will
increase the permeability of the Gram-negative outer membrane,
facilitating exposure of the inner cell wall to the lytic proteins
described in this disclosure, resulting in rapid cell lysis.
[0211] Additional embodiments include, but are not limited to,
bacteria that can be effectively lysed with the lysins described in
this disclosure include any bacterial species that contain the
chemical bonds in peptidoglycan recognized by the four classes of
lytic proteins. A review of some pathogenic bacteria genome
sequences reveals that a significant number contains at least six
to eight genes encoding the four classes of lytic proteins. Not all
lytic proteins are expected to have the same effectiveness, so
testing of the qualitative and quantitative lytic activity of a
candidate lysin is required to determine relative activity, enzyme
stability and other properties in order to identify the specific
lytic enzyme that is the best choice for a particular use.
[0212] In other embodiments, the methods described herein can be
performed to extract DNA from the new pathogen and subject the DNA
to deep sequencing to provide an unfinished genome sequence using
available DNA sequencing, sequence assembly and annotation
technologies. In these methods, it is possible to identify genes
encoding these enzymes, cloning, expression and purification of the
lytic proteins and production of this material within a short
period of time without need of identifying the pathogen.
[0213] In other embodiments, the lysins described in this
disclosure can be used to decontaminate a surface or area or room
or object contaminated with Bacillus anthracis and certain related
bacteria by contacting the surface or area or room or object with
one or more lysins described in this disclosure, such that the cell
walls of Bacillus anthracis or certain related bacteria present on
the surface or area or room or object will be lysed by the lysins
described in this disclosure and the Bacillus anthracis or certain
related bacteria effectively killed or neutralized. Examples of a
surface or area or room or object that may be decontaminated by the
lysins described in the disclosure may include, but are not limited
to machines and instruments, building areas, furniture, articles of
manufacture and personal effects, and foods. The lysins described
in the disclosure may be used to decontaminate surfaces or areas or
rooms or objects that humans contact with.
[0214] In other embodiments, the lysins described in this
disclosure may be applied to the surface or area or room or object
contaminated with Bacillus anthracis and certain related bacteria
in a form that is suitable for the decontamination of a particular
surface or area or room or object. As one non-limiting example, the
lysins may be applied as a powder form to a particular surface or
area or room or object and reconstituted with a fluid that allows
the lysins to lyse the cell walls of Bacillus anthracis or certain
related bacteria and effectively kill or neutralize them. As
another non-limiting example, the lysins may be applied as a liquid
form to a particular surface or area or room or object to lyse the
cell walls of Bacillus anthracis or certain related bacteria and
effectively kill or neutralize them.
[0215] In other embodiments, the lysins described in this
disclosure can be used to disinfect a surface or area or room or
object contaminated with at least Bacillus anthracis and certain
related bacteria by contacting the surface or area or room or
object with one or more lysins described in this disclosure, such
that the cell walls of Bacillus anthracis or certain related
bacteria present on the surface or area or room or object will be
lysed by the lysins described in this disclosure and the Bacillus
anthracis or certain related bacteria effectively killed or
neutralized. Examples of a surface or area or room or object that
may be disinfected by the lysins described in the disclosure may
include, but are not limited to machines and instruments, building
areas, furniture, articles of manufacture and personal effects, and
foods. The lysins described in the disclosure may be used to
disinfect surfaces or areas or rooms or objects that humans contact
with.
[0216] In other embodiments, the lysins described in this
disclosure may be applied to the surface or area or room or object
contaminated with at least Bacillus anthracis and certain related
bacteria in a form that is suitable for the disinfecting of a
particular surface or area or room or object. As one non-limiting
example, the lysins may be applied as a powder form to a particular
surface or area or room or object and reconstituted with a fluid
that allows the lysins to lyse the cell walls of Bacillus anthracis
or certain related bacteria and effectively kill or neutralize
them. As another non-limiting example, the lysins may be applied as
a liquid form to a particular surface or area or room or object to
lyse the cell walls of Bacillus anthracis or certain related
bacteria and effectively kill or neutralize them.
EXAMPLES
[0217] In the examples below, pBQ13 encoding BQ13 (BA_2528_wild
type), and derivatives pBQ22, encoding BQ22 (BA_2528 N-terminal
6.times.HisTag), pBQ23, encoding BQ23 (BA_2528 C-terminal
6.times.HisTag), and pBQ24 encoding BQ24 (BA_2528 Catalytic domain
only, untagged), were expressed from pET16b in E. coli BL21(DE3)
Codon Plus RIL to maximize the possibility of expression of soluble
active enzyme.
[0218] IPTG-induced whole cell lysates were analyzed by SDS
PAGE/Coomassie staining to visualize expression of a protein with
the predicted molecular weight; see, for example, FIG. 3.
[0219] Triton X-100 (TX-100)-soluble fractions from the lysates
were analyzed by SDS PAGE/Coomassie staining to visualize the
presence of the lysin candidate in the crude lysate fraction that
was analyzed for activity; see, for example, FIG. 4.
[0220] Lysins were assayed for activity by monitoring the decrease
in A600 of Bacillus anthracis Sterne at 35.degree. C. upon addition
of TX-100 extracted crude E. coli lysates of IPTG induced cells
expressing the lysin of interest using the 96-well plate format in
50 mM Tris 7.5 0.1% TX-100, which was previously shown to be
optimal for BQ13; see for example, FIG. 5. The lysins were purified
from Triton X-100-extracted E. coli lysates using IMAC; see for
example FIG. 6.
[0221] Normalized molar amounts (from 2.32E-2 nmol to 7.25E-4 nmol)
of purified lysins were tested for activity against Bacillus
anthracis Sterne and both BQ22 (BA_2528 N-terminal 6XH), and BQ23
(BA_2528 C-terminal 6XH) were active; see for example FIG. 7). Data
from the addition of 2.32E-2 nmol purified protein is shown in FIG.
7.
Example I
Example Bacterial Strains and Growth Conditions:
[0222] Escherichia coli (E. coli) strains are cultured in
Luria-Bertani broth (LB, Difco) at 28.degree. C. Strain BL21(DE3)
Codon Plus RIL (Stratagene), which lacks the outer membrane
proteases Lon and OmpT, and which has a plasmid that encodes rare
tRNAs for codons present in AT-rich organisms (such as Bacillus
anthracis), was chosen for expression of lysin proteins. Ampicillin
(Amp) is used at a concentration of 100 .mu.g/mL in plates and at
125 .mu.g/mL in liquid medium. Chloramphenicol (Cam) was used at 34
.mu.g/mL in plates and liquid medium. The concentration of
chloramphenicol was increased to 50 .mu.g/mL in overnight
cultures.
Example II
DNA Isolation and Manipulation:
[0223] Candidate lysin genes are amplified by PCR from strain
specific genomic DNA using candidate lysin (CL)-specific primers
that contain restriction sites compatible with the multi-cloning
site of pET16b. PCR products are purified using a Qiagen PCR
purification kit (Qiagen, Inc., Hilden, Germany) according to the
manufacturer's instructions, and the concentration of the amplified
DNA is determined using a Nanodrop 1000 spectrophotometer (X-Rite,
Grand Rapids, Mich.). DNA fragments encoding the genes of interest
are ligated into the pET16b(+) vector (Novagen), placing the
candidate lysin sequence under the transcriptional control of the
T7 promoter and under the translational control of the pET16b(+)
strong RBS. Plasmid pET16b was chosen for expression of lysin
proteins because it encodes lad for high level repression of
transcription prior to induction with IPTG. Ligation mixtures are
transformed to E. coli XL10 Gold (Stratagene) according to the
manufacturer's instructions and transformants are selected on
LB/amp plates. Transformants are sub-cultured for purity on to
LB/amp plates and plasmid isolations are prepared using a Qiagen
miniprep spin kit according to the manufacturer's instructions.
Plasmid DNA concentration is determined using a Nanodrop 1000
spectrophotometer. CL genes that are difficult to amplify directly
from genomic DNA are TOPO cloned or re-amplified from initial PCR
products and then cloned into pET16b using the appropriate
restriction endonucleases.
Example III
DNA Sequencing:
[0224] Primers T7PSense having SEQ ID NO: 3 (5'
cgatcccgcgaaattaatacgactcactatagg 3') and T7Tanti having SEQ ID NO:
4 (5' gctagttattgctcagcggtggc 3') are used to amplify the DNA
encoding each CL from pET16b-based plasmids. PCR reactions are
treated with ExoSAP-IT (Affymetrix) according to the manufacturer's
instructions and quantified on a 0.8% agarose minigel (Invitrogen,
Thermo Fisher Scientific., Inc, Waltham, Mass.) by estimation
relative to a known amount of the NEB 1 Kb ladder (NEB). A series
of CL-specific primers that provide at least 2.times. sequence
coverage across the sequence are used with a BigDye chain
terminator cycle sequencing kit (ABI) according to the
manufacturer's instructions. An ABI 3130 XL automated DNA sequencer
with analysis software v5.0 is used to generate sequence data.
Sequencher (Gene codes) is used to assemble sequence data. BLASTn
is used to verify correct sequence relative to the CL specific
plasmid map. Candidates with confirmed correct sequences are
transformed into E. coli BL21(DE3) Codon plus RIL for protein
expression.
Example IV
Expression of Candidate Lysins:
[0225] Strain BL BL21(DE3) Codon Plus RIL containing the plasmid of
interest is incubated overnight at 28.degree. C. in LB/Amp/Cam
without shaking. The culture is then diluted 1:500 in fresh LB with
Amp and Cam and cultured at 28.degree. C. with shaking at 120 rpm
until reaching A600 nm=0.4-0.6. IPTG is added to a final
concentration of 1 mM, and cells are cultured for a further 3 h.
Cells are pelleted by centrifugation, the supernatant is removed,
and the cell pellets flash frozen on dry ice. Cell pellets are
stored at -80.degree. C. until used in SDS PAGE or activity assays.
The induction procedure may be modified based upon the observed
characteristics of any CL when performing the initial induction as
described.
Example V
[0226] SDS-PAGE: IPTG-induced cell pellets are resuspended in
1.times.SDS sample buffer and heated to 99.degree. C. for 10 min in
a heat block. Samples are analyzed by SDS-PAGE using a Bio-Rad
4-12% TGX minigel gel (Bio-Rad, Hercules, Calif.) at 200V for 35
min in Tris-glycine SDS running buffer. Gels are then stained with
Simply Blue safe stain (Invitrogen) according to the manufacturer's
instructions. The apparent molecular mass of protein products from
IPTG induction experiments is determined by comparison to the Novex
Sharp Prestained molecular mass standard (Invitrogen).
Example VI
Activity Assay:
[0227] All operations involving wildtype Bacillus anthracis are
performed within a class IIA2 BSC in a select agent-registered
laboratory using BSL-3 procedures. Bacillus anthracis Sterne, an
avirulent select agent-excluded strain, is grown on LB plates at
35.degree. C. overnight. After approximately 16 h growth, the cells
are suspended in cation-adjusted Mueller Hinton broth with TES
(CAMHB, Difco) to an absorbance of 0.1 using a Dade-Behring
spectrophotometer, diluted 1:100 in LB and incubated for 4 h at
35.degree. C., shaking at 120 rpm in a plastic flask with 0.22
.mu.m aerator top. Cells are then captured on a 0.22 .mu.m CA
membrane using a vacuum apparatus, washed with d-H20 (Cellgro,
Manassas, Va.), resuspended in d-H20 by vigorous vortexing and held
at room temperature until used in the activity assay. All
operations for preparation of E. coli lysates are performed at
4.degree. C. unless otherwise noted. An IPTG-induced E. coli cell
pellet is thawed, re-suspended in lysis buffer (25 mM Tris 8.0),
and sonicated using a Covaris S220 adaptive focused acoustic
sonicator. A fraction of the lysate is brought to a final
concentration of 1% TX-100 and incubated 15 min. Lysates are
centrifuged at 13,000 rpm for 2 min, the supernatant is transferred
to new 1.5 mL Eppendorf tube, and a sample is removed for analysis
of induction by SDS PAGE. An aliquot of 155 .mu.l, of Bacillus
anthracis Sterne in d-H20 is added to a well of a 96-well plate
containing 20 .mu.l, of 10.times. buffer such that, after adding 25
mL of crude cell lysate, the final concentration of 50 mM buffer is
present in a 2004, assay volume. Activity is analyzed under the
following conditions, all of which may not be tested at any one
given time: 50 mM acetate pH 5.2, 50 mM IVIES pH 5.2, 50 mM
phosphate pH 6.0, 7.0, and 8.0, PBS pH 7.4, 50 mM Tris pH 6.5, 7.5,
and 8.5. Any of the given reaction conditions can be modified by
addition of NaCl, divalent cations, DTT, EDTA and Triton X-100
taking into consideration factors such as the solubility constants
of the individual buffers when combined to the cation used; the
effects of EDTA on the total concentration of metal ions such as
Zn2+; and the requirements of cofactors for enzymatic activity in
some but not all lysin proteins. The temperature may also be
varied. A 25 .mu.l, volume of crude lysate or purified protein at a
predetermined concentration is added to initiate the reaction and
bring the final volume to 200 .mu.L. Plates are covered and
incubated for 30 min in a Spectramax 384 plus (Molecular Devices)
with A600 readings taken at 1 min intervals. Positive and negative
controls are included; the positive control is IMAC-purified
BCZK2532, and the negative control is re-suspension buffer.
Example VII
Purification of 6.times.HisTag Proteins:
[0228] All operations are performed at 4.degree. C. unless
otherwise specified. An IPTG-induced E. coli cell pellet is thawed
at 4.degree. C., resuspended in lysis buffer (25 mM Tris 8.0, 300
mM NaCl) and sonicated using a Covaris S220 adaptive focused
acoustics sonicator. In the event that activity is detected in the
TX-100 soluble fraction of the crude lysate activity assay as
described above, Triton X-100 is added to a final concentration of
1% and the cell lysate is extracted for 30 min. Lysates are
centrifuged at 4000 rpm for 10 min. The supernatant is transferred
to a new tube containing HisPur Ni-NTA Resin (Thermo), and binding
is conducted for 30 min. The Ni-NTA resin is washed, and the
protein of interest is eluted from the resin according to the
manufacturer's instructions.
[0229] The contents of all references cited in the present
specifications and all cited references in each of those references
are incorporated in their entirety by reference herein.
[0230] While many embodiments have been disclosed above, many other
embodiments and variations are possible within the scope of the
present disclosure as recognized by one of ordinary skill in the
art and in the appended claims that follow. Accordingly, the
details of the embodiments and examples provided are not to be
construed as limiting. It is to be understood that the terms used
herein are merely descriptive rather than limiting and that various
changes and numerous equivalents may be made without departing from
the spirit or scope of the claimed invention.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 12 <210> SEQ ID NO 1 <211> LENGTH: 1454
<212> TYPE: DNA <213> ORGANISM: artificial <220>
FEATURE: <223> OTHER INFORMATION: BQ22 lysin protein gene
construct <400> SEQUENCE: 1 cggtgatgcc ggccacgatg cgtccggcgt
agaggatcga gatctcgatc ccgcgaaatt 60 aatacgactc actatagggg
aattgtgagc ggataacaat tcccctctag aaataatttt 120 gtttaacttt
aagaaggaga tataccatgg gccatcatca tcatcatcat agcgcagata 180
ctcacagatt cccagatgtt cctgcatggg ctgacaaatc cgttacttat ttagttgata
240 aacaagtatt gagtggttat ccagatggga cttttggttc aagtgataca
ctagatagag 300 cttctgcagc aacaattatg actaaggctc ttggtataca
cattgattta aatgcaaaac 360 catcttttaa agattcacaa aaccactggg
gaacccctta tattgccgca gctgaaaagg 420 caggaatcat taaaggtgaa
ggaaatggaa tatttaatcc ttctggaaaa gttactcgtg 480 ctgctatggc
tactatgcta gtgaatgcat ataaactaca aaataaaaac actagcaatg 540
gacaaagtaa atttgaagat ttaaagggcc attggggtga aaagttcgca aatactttaa
600 ttgatttgaa aatttcagtt ggtacagata atggctggca accaaataaa
ttcataacac 660 gcgctgaagc tgcacaacta actgcaaaaa cagatatgct
tcaatatagt catagtaatc 720 ctttagaaaa taaaaccata attattgatc
ccggacatgg tggcgaagat cctggaaaag 780 acacaaaggg attacctgaa
agtaagattg tactagacac ttctttacgt ctacaaaaat 840 tgcttgaaaa
acatacacca tttacagttt tactaactcg taaatctgat actagaccag 900
gacatgatca aaaaagctct ttacaggaac gtgtgaaatt tgctaaacaa aatcaggggg
960 atatctttat aagtgttcat gcaaatgctt ttaatggtaa tgcaaaaggg
acggaaacat 1020 actactataa atcttctaaa tctgaaaaaa caaatcctca
tgtggaagag agtcgtgttt 1080 tagctgaaaa aattcaaact cgattagtag
acgctcttca aacacgtgat agaggcgtta 1140 aacatggaga tcttcatgtt
ataagagaaa atgacatgcc agctgtgtta acggaacttg 1200 cttttataga
taatggtatc gattacagta agttatctac agaaaacgga aggcagattg 1260
ctgcagaagc catttatgag gggattttag attattatga atggaaagga aataatgtat
1320 ctgaatatag gctgtaactc gaggatccgg ctgctaacaa agcccgaaag
gaagctgagt 1380 tggctgctgc caccgctgag caataactag cataacccct
tggggcctct aaacgggtct 1440 tgaggggttt tttg 1454 <210> SEQ ID
NO 2 <211> LENGTH: 396 <212> TYPE: PRT <213>
ORGANISM: artificial <220> FEATURE: <223> OTHER
INFORMATION: BQ22 lysin protein <400> SEQUENCE: 2 Met Gly His
His His His His His Ser Ala Asp Thr His Arg Phe Pro 1 5 10 15 Asp
Val Pro Ala Trp Ala Asp Lys Ser Val Thr Tyr Leu Val Asp Lys 20 25
30 Gln Val Leu Ser Gly Tyr Pro Asp Gly Thr Phe Gly Ser Ser Asp Thr
35 40 45 Leu Asp Arg Ala Ser Ala Ala Thr Ile Met Thr Lys Ala Leu
Gly Ile 50 55 60 His Ile Asp Leu Asn Ala Lys Pro Ser Phe Lys Asp
Ser Gln Asn His 65 70 75 80 Trp Gly Thr Pro Tyr Ile Ala Ala Ala Glu
Lys Ala Gly Ile Ile Lys 85 90 95 Gly Glu Gly Asn Gly Ile Phe Asn
Pro Ser Gly Lys Val Thr Arg Ala 100 105 110 Ala Met Ala Thr Met Leu
Val Asn Ala Tyr Lys Leu Gln Asn Lys Asn 115 120 125 Thr Ser Asn Gly
Gln Ser Lys Phe Glu Asp Leu Lys Gly His Trp Gly 130 135 140 Glu Lys
Phe Ala Asn Thr Leu Ile Asp Leu Lys Ile Ser Val Gly Thr 145 150 155
160 Asp Asn Gly Trp Gln Pro Asn Lys Phe Ile Thr Arg Ala Glu Ala Ala
165 170 175 Gln Leu Thr Ala Lys Thr Asp Met Leu Gln Tyr Ser His Ser
Asn Pro 180 185 190 Leu Glu Asn Lys Thr Ile Ile Ile Asp Pro Gly His
Gly Gly Glu Asp 195 200 205 Pro Gly Lys Asp Thr Lys Gly Leu Pro Glu
Ser Lys Ile Val Leu Asp 210 215 220 Thr Ser Leu Arg Leu Gln Lys Leu
Leu Glu Lys His Thr Pro Phe Thr 225 230 235 240 Val Leu Leu Thr Arg
Lys Ser Asp Thr Arg Pro Gly His Asp Gln Lys 245 250 255 Ser Ser Leu
Gln Glu Arg Val Lys Phe Ala Lys Gln Asn Gln Gly Asp 260 265 270 Ile
Phe Ile Ser Val His Ala Asn Ala Phe Asn Gly Asn Ala Lys Gly 275 280
285 Thr Glu Thr Tyr Tyr Tyr Lys Ser Ser Lys Ser Glu Lys Thr Asn Pro
290 295 300 His Val Glu Glu Ser Arg Val Leu Ala Glu Lys Ile Gln Thr
Arg Leu 305 310 315 320 Val Asp Ala Leu Gln Thr Arg Asp Arg Gly Val
Lys His Gly Asp Leu 325 330 335 His Val Ile Arg Glu Asn Asp Met Pro
Ala Val Leu Thr Glu Leu Ala 340 345 350 Phe Ile Asp Asn Gly Ile Asp
Tyr Ser Lys Leu Ser Thr Glu Asn Gly 355 360 365 Arg Gln Ile Ala Ala
Glu Ala Ile Tyr Glu Gly Ile Leu Asp Tyr Tyr 370 375 380 Glu Trp Lys
Gly Asn Asn Val Ser Glu Tyr Arg Leu 385 390 395 <210> SEQ ID
NO 3 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic primer - T7PSense <400> SEQUENCE: 3
cgatcccgcg aaattaatac gactcactat agg 33 <210> SEQ ID NO 4
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic primer - T7Tanti <400> SEQUENCE: 4 gctagttatt
gctcagcggt ggc 23 <210> SEQ ID NO 5 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: artificial <220>
FEATURE: <223> OTHER INFORMATION: His-Tag sequence
<400> SEQUENCE: 5 catcatcatc atcatcat 18 <210> SEQ ID
NO 6 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: artificial <220> FEATURE: <223> OTHER
INFORMATION: Lac operator <400> SEQUENCE: 6 gggaattgtg
agcggataac aatt 24 <210> SEQ ID NO 7 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: artificial <220>
FEATURE: <223> OTHER INFORMATION: T7 promoter <400>
SEQUENCE: 7 taatacgact cactataggg 20 <210> SEQ ID NO 8
<211> LENGTH: 1165 <212> TYPE: DNA <213>
ORGANISM: Bacillus anthracis <400> SEQUENCE: 8 cgcagatact
cacagattcc cagatgttcc tgcatgggct gacaaatccg ttacttattt 60
agttgataaa caagtattga gtggttatcc agatgggact tttggttcaa gtgatacact
120 agatagagct tctgcagcaa caattatgac taaggctctt ggtatacaca
ttgatttaaa 180 tgcaaaacca tcttttaaag attcacaaaa ccactgggga
accccttata ttgccgcagc 240 tgaaaaggca ggaatcatta aaggtgaagg
aaatggaata tttaatcctt ctggaaaagt 300 tactcgtgct gctatggcta
ctatgctagt gaatgcatat aaactacaaa ataaaaacac 360 tagcaatgga
caaagtaaat ttgaagattt aaagggccat tggggtgaaa agttcgcaaa 420
tactttaatt gatttgaaaa tttcagttgg tacagataat ggctggcaac caaataaatt
480 cataacacgc gctgaagctg cacaactaac tgcaaaaaca gatatgcttc
aatatagtca 540 tagtaatcct ttagaaaata aaaccataat tattgatccc
ggacatggtg gcgaagatcc 600 tggaaaagac acaaagggat tacctgaaag
taagattgta ctagacactt ctttacgtct 660 acaaaaattg cttgaaaaac
atacaccatt tacagtttta ctaactcgta aatctgatac 720 tagaccagga
catgatcaaa aaagctcttt acaggaacgt gtgaaatttg ctaaacaaaa 780
tcagggggat atctttataa gtgttcatgc aaatgctttt aatggtaatg caaaagggac
840 ggaaacatac tactataaat cttctaaatc tgaaaaaaca aatcctcatg
tggaagagag 900 tcgtgtttta gctgaaaaaa ttcaaactcg attagtagac
gctcttcaaa cacgtgatag 960 aggcgttaaa catggagatc ttcatgttat
aagagaaaat gacatgccag ctgtgttaac 1020 ggaacttgct tttatagata
atggtatcga ttacagtaag ttatctacag aaaacggaag 1080 gcagattgct
gcagaagcca tttatgaggg gattttagat tattatgaat ggaaaggaaa 1140
taatgtatct gaatataggc tgtaa 1165 <210> SEQ ID NO 9
<211> LENGTH: 387 <212> TYPE: PRT <213> ORGANISM:
Bacillus anthracis <400> SEQUENCE: 9 Ala Asp Thr His Arg Phe
Pro Asp Val Pro Ala Trp Ala Asp Lys Ser 1 5 10 15 Val Thr Tyr Leu
Val Asp Lys Gln Val Leu Ser Gly Tyr Pro Asp Gly 20 25 30 Thr Phe
Gly Ser Ser Asp Thr Leu Asp Arg Ala Ser Ala Ala Thr Ile 35 40 45
Met Thr Lys Ala Leu Gly Ile His Ile Asp Leu Asn Ala Lys Pro Ser 50
55 60 Phe Lys Asp Ser Gln Asn His Trp Gly Thr Pro Tyr Ile Ala Ala
Ala 65 70 75 80 Glu Lys Ala Gly Ile Ile Lys Gly Glu Gly Asn Gly Ile
Phe Asn Pro 85 90 95 Ser Gly Lys Val Thr Arg Ala Ala Met Ala Thr
Met Leu Val Asn Ala 100 105 110 Tyr Lys Leu Gln Asn Lys Asn Thr Ser
Asn Gly Gln Ser Lys Phe Glu 115 120 125 Asp Leu Lys Gly His Trp Gly
Glu Lys Phe Ala Asn Thr Leu Ile Asp 130 135 140 Leu Lys Ile Ser Val
Gly Thr Asp Asn Gly Trp Gln Pro Asn Lys Phe 145 150 155 160 Ile Thr
Arg Ala Glu Ala Ala Gln Leu Thr Ala Lys Thr Asp Met Leu 165 170 175
Gln Tyr Ser His Ser Asn Pro Leu Glu Asn Lys Thr Ile Ile Ile Asp 180
185 190 Pro Gly His Gly Gly Glu Asp Pro Gly Lys Asp Thr Lys Gly Leu
Pro 195 200 205 Glu Ser Lys Ile Val Leu Asp Thr Ser Leu Arg Leu Gln
Lys Leu Leu 210 215 220 Glu Lys His Thr Pro Phe Thr Val Leu Leu Thr
Arg Lys Ser Asp Thr 225 230 235 240 Arg Pro Gly His Asp Gln Lys Ser
Ser Leu Gln Glu Arg Val Lys Phe 245 250 255 Ala Lys Gln Asn Gln Gly
Asp Ile Phe Ile Ser Val His Ala Asn Ala 260 265 270 Phe Asn Gly Asn
Ala Lys Gly Thr Glu Thr Tyr Tyr Tyr Lys Ser Ser 275 280 285 Lys Ser
Glu Lys Thr Asn Pro His Val Glu Glu Ser Arg Val Leu Ala 290 295 300
Glu Lys Ile Gln Thr Arg Leu Val Asp Ala Leu Gln Thr Arg Asp Arg 305
310 315 320 Gly Val Lys His Gly Asp Leu His Val Ile Arg Glu Asn Asp
Met Pro 325 330 335 Ala Val Leu Thr Glu Leu Ala Phe Ile Asp Asn Gly
Ile Asp Tyr Ser 340 345 350 Lys Leu Ser Thr Glu Asn Gly Arg Gln Ile
Ala Ala Glu Ala Ile Tyr 355 360 365 Glu Gly Ile Leu Asp Tyr Tyr Glu
Trp Lys Gly Asn Asn Val Ser Glu 370 375 380 Tyr Arg Leu 385
<210> SEQ ID NO 10 <211> LENGTH: 410 <212> TYPE:
PRT <213> ORGANISM: Bacillus anthracis <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
NCBI/NP_844897 <309> DATABASE ENTRY DATE: 2017-10-06
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(410) <400>
SEQUENCE: 10 Met Lys Asn Lys Leu Ile Ala Thr Gly Ile Leu Ala Gly
Ser Leu Leu 1 5 10 15 Ser Tyr Ser Thr Ser Ile Val Ala Asp Thr His
Arg Phe Pro Asp Val 20 25 30 Pro Ala Trp Ala Asp Lys Ser Val Thr
Tyr Leu Val Asp Lys Gln Val 35 40 45 Leu Ser Gly Tyr Pro Asp Gly
Thr Phe Gly Ser Ser Asp Thr Leu Asp 50 55 60 Arg Ala Ser Ala Ala
Thr Ile Met Thr Lys Ala Leu Gly Ile His Ile 65 70 75 80 Asp Leu Asn
Ala Lys Pro Ser Phe Lys Asp Ser Gln Asn His Trp Gly 85 90 95 Thr
Pro Tyr Ile Ala Ala Ala Glu Lys Ala Gly Ile Ile Lys Gly Glu 100 105
110 Gly Asn Gly Ile Phe Asn Pro Ser Gly Lys Val Thr Arg Ala Ala Met
115 120 125 Ala Thr Met Leu Val Asn Ala Tyr Lys Leu Gln Asn Lys Asn
Thr Ser 130 135 140 Asn Gly Gln Ser Lys Phe Glu Asp Leu Lys Gly His
Trp Gly Glu Lys 145 150 155 160 Phe Ala Asn Thr Leu Ile Asp Leu Lys
Ile Ser Val Gly Thr Asp Asn 165 170 175 Gly Trp Gln Pro Asn Lys Phe
Ile Thr Arg Ala Glu Ala Ala Gln Leu 180 185 190 Thr Ala Lys Thr Asp
Met Leu Gln Tyr Ser His Ser Asn Pro Leu Glu 195 200 205 Asn Lys Thr
Ile Ile Ile Asp Pro Gly His Gly Gly Glu Asp Pro Gly 210 215 220 Lys
Asp Thr Lys Gly Leu Pro Glu Ser Lys Ile Val Leu Asp Thr Ser 225 230
235 240 Leu Arg Leu Gln Lys Leu Leu Glu Lys His Thr Pro Phe Thr Val
Leu 245 250 255 Leu Thr Arg Lys Ser Asp Thr Arg Pro Gly His Asp Gln
Lys Ser Ser 260 265 270 Leu Gln Glu Arg Val Lys Phe Ala Lys Gln Asn
Gln Gly Asp Ile Phe 275 280 285 Ile Ser Val His Ala Asn Ala Phe Asn
Gly Asn Ala Lys Gly Thr Glu 290 295 300 Thr Tyr Tyr Tyr Lys Ser Ser
Lys Ser Glu Lys Thr Asn Pro His Val 305 310 315 320 Glu Glu Ser Arg
Val Leu Ala Glu Lys Ile Gln Thr Arg Leu Val Asp 325 330 335 Ala Leu
Gln Thr Arg Asp Arg Gly Val Lys His Gly Asp Leu His Val 340 345 350
Ile Arg Glu Asn Asp Met Pro Ala Val Leu Thr Glu Leu Ala Phe Ile 355
360 365 Asp Asn Gly Ile Asp Tyr Ser Lys Leu Ser Thr Glu Asn Gly Arg
Gln 370 375 380 Ile Ala Ala Glu Ala Ile Tyr Glu Gly Ile Leu Asp Tyr
Tyr Glu Trp 385 390 395 400 Lys Gly Asn Asn Val Ser Glu Tyr Arg Leu
405 410 <210> SEQ ID NO 11 <211> LENGTH: 348
<212> TYPE: PRT <213> ORGANISM: Bacillus
weihenstephanensis <300> PUBLICATION INFORMATION: <308>
DATABASE ACCESSION NUMBER: NCBI/YP_001646166.1 <309> DATABASE
ENTRY DATE: 2014-12-16 <313> RELEVANT RESIDUES IN SEQ ID NO:
(1)..(348) <400> SEQUENCE: 11 Met Gln Asn Arg Ser Ser Ser Asn
Ile Thr Phe Ile Asp Val Ser His 1 5 10 15 Trp Glu Gly Asn Ile Asn
Trp Asn Ala Val Lys Ser Ser Gly Ile Pro 20 25 30 Ala Ala Tyr Ala
Lys Ala Thr Glu Gly Val Asn Tyr Ile Asp Pro Thr 35 40 45 Phe Val
Gln Asn Val Gln Ala Ala Arg Asn Ala Asn Val Leu Ile Gly 50 55 60
Ala Tyr His Phe Ala His Pro Glu Gln Asn Asp Ala Ile Ser Glu Ala 65
70 75 80 Lys Tyr Phe Val Ser Ile Leu Gln Ser Asn Gln Thr Asp Leu
Ile Pro 85 90 95 Val Leu Asp Leu Glu Ser Pro Thr Asp Thr Ser Asn
Ser Ser Leu Thr 100 105 110 Gly Ala Thr Ile Ser Asn Trp Ala Arg Ser
Phe Val Asn Tyr Val Lys 115 120 125 Gln Ala Thr Gly Lys Asp Val Met
Leu Tyr Thr Gly Ile Trp Tyr Ile 130 135 140 Asn Glu Phe Gly Ile Ser
Gly Leu Ser Asp Ile Pro Leu Trp Ile Ser 145 150 155 160 Lys Tyr Ser
Ser Ile Pro Pro Ala Asp Ala Gly Gly Trp Thr Glu Trp 165 170 175 Thr
Ala Trp Gln Tyr Thr Asp Ser Gly Gln Ile Ser Gly Val Gly Asn 180 185
190 Cys Asp Val Ser Ala Ala Val Ser Leu Glu Ala Leu Gln Gly Asn Gly
195 200 205 Ala Ser Gly Gly Gly Asn Val Ser Thr Pro Asn Asn Ala Thr
Pro Val 210 215 220 Tyr Gly Val Ala Val Ile Asn Gly Asp Asn Val Asn
Leu Arg Ser Gly 225 230 235 240 Pro Ser Leu Gln Ser Ser Val Ile Arg
Gln Leu Asn Arg Gly Glu Ser 245 250 255 Tyr Glu Val Trp Gly Glu Gln
Asn Gly Trp Leu Cys Leu Gly Thr Asn 260 265 270 Gln Trp Val Tyr Asn
Asp Ser Ser Tyr Ile Gln Tyr Lys His Tyr Val 275 280 285 Ala Thr Ile
Thr Gly Asp Asn Val Asn Leu Arg Asp Ala Pro Ser Leu 290 295 300 Asn
Gly Asn Val Ile Arg Gln Leu His His Gly Glu Ser Tyr Arg Val 305 310
315 320 Trp Ser Lys Gln Asp Gly Trp Leu Cys Leu Gly Thr Asn Gln Trp
Val 325 330 335 Tyr Tyr Asp Ser Ser Tyr Ile Gln Tyr Gly Val Gln 340
345 <210> SEQ ID NO 12 <211> LENGTH: 529 <212>
TYPE: PRT <213> ORGANISM: Bacillus anthracis <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
NCBI/NP_843409 <309> DATABASE ENTRY DATE: 2017-10-06
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(529) <400>
SEQUENCE: 12 Met Lys Tyr Arg Ala Val Ala Ala Gly Ile Leu Ala Ala
Ser Leu Leu 1 5 10 15 Ser Ser Pro Val Ser Ser Phe Ala Ala Ala Lys
Lys Phe Ser Asp Val 20 25 30 Pro Thr Trp Ala Gln Gln Ser Val Asp
Tyr Leu Val Gly Lys Lys Ala 35 40 45 Leu Asp Gly Lys Pro Asp Gly
Thr Phe Ser Pro Ser Glu Ala Val Asp 50 55 60 Arg Gly Ser Ala Ala
Lys Ile Leu Ala Val Val Leu Gly Leu Pro Val 65 70 75 80 Asp Pro Lys
Ala Lys Pro Ser Phe Lys Asp Ala Gln Asn His Trp Ala 85 90 95 Ala
Pro Tyr Ile Ala Ala Val Glu Lys Ala Gly Val Ile Asn Gly Asp 100 105
110 Gly Thr Gly Lys Phe Asn Pro Ser Ser Gln Ile Asn Arg Ala Ser Met
115 120 125 Ala Ser Met Leu Val Gln Ala Tyr Ser Leu Asp Lys Lys Ile
Ile Gly 130 135 140 Glu Leu Pro Thr Gln Phe Lys Asp Leu Glu Pro His
Trp Gly Lys Lys 145 150 155 160 Gln Ala Asn Ile Leu Val Ala Leu Glu
Ile Ser Lys Gly Thr Gly Asn 165 170 175 Gly Trp Asn Pro Glu Gly Thr
Val Thr Arg Ala Glu Ala Ala Gln Phe 180 185 190 Ile Ala Met Ala Asp
Gln Asn Lys Thr Ser Thr Ser Lys Arg Met Tyr 195 200 205 Met Asn Arg
Asn Val Ile Thr Tyr His Gln Pro Ser Leu Ser Ser Gly 210 215 220 Ile
Thr Asp Val Gln His Lys Pro Gln Met Val Glu Val Thr Glu Gln 225 230
235 240 Arg Ala Asp Gly Trp Leu Lys Ile Val Thr Ser Lys Gly Glu Lys
Trp 245 250 255 Thr Pro Leu Thr Glu Lys Thr Glu Thr Ile Asn Glu Glu
Phe Thr Thr 260 265 270 Tyr Glu Thr Ala Ser His Ser Ser Lys Val Leu
Gly Thr Tyr Asn Ala 275 280 285 Gln Thr Val Thr Val Met Glu Glu Ser
Gly Ser Trp Ile Arg Ile Arg 290 295 300 Val Gly Ala Gly Phe Gln Trp
Val Asp Lys Asn Gln Leu Asn Pro Val 305 310 315 320 Lys Gln Glu Asn
Phe Leu Glu Gly Lys Ala Ile Ile Ile Asp Pro Gly 325 330 335 His Gly
Gly Ile Asp Ser Gly Asn Val Gly Tyr Tyr Glu Lys Glu Ser 340 345 350
Glu Thr Val Leu Asp Val Ser Leu Arg Leu Lys Lys Ile Phe Glu Gln 355
360 365 Lys Ala Pro Phe Thr Val Met Phe Thr Arg Thr Asp Asn Thr Arg
Pro 370 375 380 Gly Val Asn Ser Thr Asp Ser Leu Lys Lys Arg Val Glu
Phe Ala Gln 385 390 395 400 Glu His Asn Gly Asp Ile Phe Val Ser Ile
His Ala Asn Gly Ser Ala 405 410 415 Glu Lys Asn Gly Gln Gly Thr Glu
Thr Leu Tyr Tyr Gln Ser Ala Arg 420 425 430 Ala Lys Val Thr Asn Pro
His Val Glu Asp Ser Lys Leu Leu Ala Gln 435 440 445 Lys Ile Gln Asp
Arg Leu Val Ala Ala Leu Gly Thr Lys Asp Arg Gly 450 455 460 Val Lys
His Gln Asp Leu Tyr Val Thr Arg Glu Asn Thr Met Pro Ala 465 470 475
480 Val Leu Thr Glu Leu Ala Phe Val Asp Asn Lys Ser Asp Ala Asp Lys
485 490 495 Ile Ala Thr Pro Lys Gln Arg Gln Ala Ala Ala Glu Ala Ile
Tyr Gln 500 505 510 Gly Ile Leu Asp Tyr Tyr Glu Ala Lys Gly Asn Asn
Val Ser Ser Phe 515 520 525 Arg
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 12 <210>
SEQ ID NO 1 <211> LENGTH: 1454 <212> TYPE: DNA
<213> ORGANISM: artificial <220> FEATURE: <223>
OTHER INFORMATION: BQ22 lysin protein gene construct <400>
SEQUENCE: 1 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
ccgcgaaatt 60 aatacgactc actatagggg aattgtgagc ggataacaat
tcccctctag aaataatttt 120 gtttaacttt aagaaggaga tataccatgg
gccatcatca tcatcatcat agcgcagata 180 ctcacagatt cccagatgtt
cctgcatggg ctgacaaatc cgttacttat ttagttgata 240 aacaagtatt
gagtggttat ccagatggga cttttggttc aagtgataca ctagatagag 300
cttctgcagc aacaattatg actaaggctc ttggtataca cattgattta aatgcaaaac
360 catcttttaa agattcacaa aaccactggg gaacccctta tattgccgca
gctgaaaagg 420 caggaatcat taaaggtgaa ggaaatggaa tatttaatcc
ttctggaaaa gttactcgtg 480 ctgctatggc tactatgcta gtgaatgcat
ataaactaca aaataaaaac actagcaatg 540 gacaaagtaa atttgaagat
ttaaagggcc attggggtga aaagttcgca aatactttaa 600 ttgatttgaa
aatttcagtt ggtacagata atggctggca accaaataaa ttcataacac 660
gcgctgaagc tgcacaacta actgcaaaaa cagatatgct tcaatatagt catagtaatc
720 ctttagaaaa taaaaccata attattgatc ccggacatgg tggcgaagat
cctggaaaag 780 acacaaaggg attacctgaa agtaagattg tactagacac
ttctttacgt ctacaaaaat 840 tgcttgaaaa acatacacca tttacagttt
tactaactcg taaatctgat actagaccag 900 gacatgatca aaaaagctct
ttacaggaac gtgtgaaatt tgctaaacaa aatcaggggg 960 atatctttat
aagtgttcat gcaaatgctt ttaatggtaa tgcaaaaggg acggaaacat 1020
actactataa atcttctaaa tctgaaaaaa caaatcctca tgtggaagag agtcgtgttt
1080 tagctgaaaa aattcaaact cgattagtag acgctcttca aacacgtgat
agaggcgtta 1140 aacatggaga tcttcatgtt ataagagaaa atgacatgcc
agctgtgtta acggaacttg 1200 cttttataga taatggtatc gattacagta
agttatctac agaaaacgga aggcagattg 1260 ctgcagaagc catttatgag
gggattttag attattatga atggaaagga aataatgtat 1320 ctgaatatag
gctgtaactc gaggatccgg ctgctaacaa agcccgaaag gaagctgagt 1380
tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
1440 tgaggggttt tttg 1454 <210> SEQ ID NO 2 <211>
LENGTH: 396 <212> TYPE: PRT <213> ORGANISM: artificial
<220> FEATURE: <223> OTHER INFORMATION: BQ22 lysin
protein <400> SEQUENCE: 2 Met Gly His His His His His His Ser
Ala Asp Thr His Arg Phe Pro 1 5 10 15 Asp Val Pro Ala Trp Ala Asp
Lys Ser Val Thr Tyr Leu Val Asp Lys 20 25 30 Gln Val Leu Ser Gly
Tyr Pro Asp Gly Thr Phe Gly Ser Ser Asp Thr 35 40 45 Leu Asp Arg
Ala Ser Ala Ala Thr Ile Met Thr Lys Ala Leu Gly Ile 50 55 60 His
Ile Asp Leu Asn Ala Lys Pro Ser Phe Lys Asp Ser Gln Asn His 65 70
75 80 Trp Gly Thr Pro Tyr Ile Ala Ala Ala Glu Lys Ala Gly Ile Ile
Lys 85 90 95 Gly Glu Gly Asn Gly Ile Phe Asn Pro Ser Gly Lys Val
Thr Arg Ala 100 105 110 Ala Met Ala Thr Met Leu Val Asn Ala Tyr Lys
Leu Gln Asn Lys Asn 115 120 125 Thr Ser Asn Gly Gln Ser Lys Phe Glu
Asp Leu Lys Gly His Trp Gly 130 135 140 Glu Lys Phe Ala Asn Thr Leu
Ile Asp Leu Lys Ile Ser Val Gly Thr 145 150 155 160 Asp Asn Gly Trp
Gln Pro Asn Lys Phe Ile Thr Arg Ala Glu Ala Ala 165 170 175 Gln Leu
Thr Ala Lys Thr Asp Met Leu Gln Tyr Ser His Ser Asn Pro 180 185 190
Leu Glu Asn Lys Thr Ile Ile Ile Asp Pro Gly His Gly Gly Glu Asp 195
200 205 Pro Gly Lys Asp Thr Lys Gly Leu Pro Glu Ser Lys Ile Val Leu
Asp 210 215 220 Thr Ser Leu Arg Leu Gln Lys Leu Leu Glu Lys His Thr
Pro Phe Thr 225 230 235 240 Val Leu Leu Thr Arg Lys Ser Asp Thr Arg
Pro Gly His Asp Gln Lys 245 250 255 Ser Ser Leu Gln Glu Arg Val Lys
Phe Ala Lys Gln Asn Gln Gly Asp 260 265 270 Ile Phe Ile Ser Val His
Ala Asn Ala Phe Asn Gly Asn Ala Lys Gly 275 280 285 Thr Glu Thr Tyr
Tyr Tyr Lys Ser Ser Lys Ser Glu Lys Thr Asn Pro 290 295 300 His Val
Glu Glu Ser Arg Val Leu Ala Glu Lys Ile Gln Thr Arg Leu 305 310 315
320 Val Asp Ala Leu Gln Thr Arg Asp Arg Gly Val Lys His Gly Asp Leu
325 330 335 His Val Ile Arg Glu Asn Asp Met Pro Ala Val Leu Thr Glu
Leu Ala 340 345 350 Phe Ile Asp Asn Gly Ile Asp Tyr Ser Lys Leu Ser
Thr Glu Asn Gly 355 360 365 Arg Gln Ile Ala Ala Glu Ala Ile Tyr Glu
Gly Ile Leu Asp Tyr Tyr 370 375 380 Glu Trp Lys Gly Asn Asn Val Ser
Glu Tyr Arg Leu 385 390 395 <210> SEQ ID NO 3 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
primer - T7PSense <400> SEQUENCE: 3 cgatcccgcg aaattaatac
gactcactat agg 33 <210> SEQ ID NO 4 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic primer - T7Tanti
<400> SEQUENCE: 4 gctagttatt gctcagcggt ggc 23 <210>
SEQ ID NO 5 <211> LENGTH: 18 <212> TYPE: DNA
<213> ORGANISM: artificial <220> FEATURE: <223>
OTHER INFORMATION: His-Tag sequence <400> SEQUENCE: 5
catcatcatc atcatcat 18 <210> SEQ ID NO 6 <211> LENGTH:
24 <212> TYPE: DNA <213> ORGANISM: artificial
<220> FEATURE: <223> OTHER INFORMATION: Lac operator
<400> SEQUENCE: 6 gggaattgtg agcggataac aatt 24 <210>
SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: artificial <220> FEATURE: <223>
OTHER INFORMATION: T7 promoter <400> SEQUENCE: 7 taatacgact
cactataggg 20 <210> SEQ ID NO 8 <211> LENGTH: 1165
<212> TYPE: DNA <213> ORGANISM: Bacillus anthracis
<400> SEQUENCE: 8 cgcagatact cacagattcc cagatgttcc tgcatgggct
gacaaatccg ttacttattt 60 agttgataaa caagtattga gtggttatcc
agatgggact tttggttcaa gtgatacact 120 agatagagct tctgcagcaa
caattatgac taaggctctt ggtatacaca ttgatttaaa 180 tgcaaaacca
tcttttaaag attcacaaaa ccactgggga accccttata ttgccgcagc 240
tgaaaaggca ggaatcatta aaggtgaagg aaatggaata tttaatcctt ctggaaaagt
300 tactcgtgct gctatggcta ctatgctagt gaatgcatat aaactacaaa
ataaaaacac 360 tagcaatgga caaagtaaat ttgaagattt aaagggccat
tggggtgaaa agttcgcaaa 420 tactttaatt gatttgaaaa tttcagttgg
tacagataat ggctggcaac caaataaatt 480 cataacacgc gctgaagctg
cacaactaac tgcaaaaaca gatatgcttc aatatagtca 540 tagtaatcct
ttagaaaata aaaccataat tattgatccc ggacatggtg gcgaagatcc 600
tggaaaagac acaaagggat tacctgaaag taagattgta ctagacactt ctttacgtct
660 acaaaaattg cttgaaaaac atacaccatt tacagtttta ctaactcgta
aatctgatac 720 tagaccagga catgatcaaa aaagctcttt acaggaacgt
gtgaaatttg ctaaacaaaa 780 tcagggggat atctttataa gtgttcatgc
aaatgctttt aatggtaatg caaaagggac 840 ggaaacatac tactataaat
cttctaaatc tgaaaaaaca aatcctcatg tggaagagag 900 tcgtgtttta
gctgaaaaaa ttcaaactcg attagtagac gctcttcaaa cacgtgatag 960
aggcgttaaa catggagatc ttcatgttat aagagaaaat gacatgccag ctgtgttaac
1020 ggaacttgct tttatagata atggtatcga ttacagtaag ttatctacag
aaaacggaag 1080 gcagattgct gcagaagcca tttatgaggg gattttagat
tattatgaat ggaaaggaaa 1140 taatgtatct gaatataggc tgtaa 1165
<210> SEQ ID NO 9 <211> LENGTH: 387 <212> TYPE:
PRT <213> ORGANISM: Bacillus anthracis <400> SEQUENCE:
9 Ala Asp Thr His Arg Phe Pro Asp Val Pro Ala Trp Ala Asp Lys Ser 1
5 10 15 Val Thr Tyr Leu Val Asp Lys Gln Val Leu Ser Gly Tyr Pro Asp
Gly 20 25 30 Thr Phe Gly Ser Ser Asp Thr Leu Asp Arg Ala Ser Ala
Ala Thr Ile 35 40 45 Met Thr Lys Ala Leu Gly Ile His Ile Asp Leu
Asn Ala Lys Pro Ser 50 55 60 Phe Lys Asp Ser Gln Asn His Trp Gly
Thr Pro Tyr Ile Ala Ala Ala 65 70 75 80 Glu Lys Ala Gly Ile Ile Lys
Gly Glu Gly Asn Gly Ile Phe Asn Pro 85 90 95 Ser Gly Lys Val Thr
Arg Ala Ala Met Ala Thr Met Leu Val Asn Ala 100 105 110 Tyr Lys Leu
Gln Asn Lys Asn Thr Ser Asn Gly Gln Ser Lys Phe Glu 115 120 125 Asp
Leu Lys Gly His Trp Gly Glu Lys Phe Ala Asn Thr Leu Ile Asp 130 135
140 Leu Lys Ile Ser Val Gly Thr Asp Asn Gly Trp Gln Pro Asn Lys Phe
145 150 155 160 Ile Thr Arg Ala Glu Ala Ala Gln Leu Thr Ala Lys Thr
Asp Met Leu 165 170 175 Gln Tyr Ser His Ser Asn Pro Leu Glu Asn Lys
Thr Ile Ile Ile Asp 180 185 190 Pro Gly His Gly Gly Glu Asp Pro Gly
Lys Asp Thr Lys Gly Leu Pro 195 200 205 Glu Ser Lys Ile Val Leu Asp
Thr Ser Leu Arg Leu Gln Lys Leu Leu 210 215 220 Glu Lys His Thr Pro
Phe Thr Val Leu Leu Thr Arg Lys Ser Asp Thr 225 230 235 240 Arg Pro
Gly His Asp Gln Lys Ser Ser Leu Gln Glu Arg Val Lys Phe 245 250 255
Ala Lys Gln Asn Gln Gly Asp Ile Phe Ile Ser Val His Ala Asn Ala 260
265 270 Phe Asn Gly Asn Ala Lys Gly Thr Glu Thr Tyr Tyr Tyr Lys Ser
Ser 275 280 285 Lys Ser Glu Lys Thr Asn Pro His Val Glu Glu Ser Arg
Val Leu Ala 290 295 300 Glu Lys Ile Gln Thr Arg Leu Val Asp Ala Leu
Gln Thr Arg Asp Arg 305 310 315 320 Gly Val Lys His Gly Asp Leu His
Val Ile Arg Glu Asn Asp Met Pro 325 330 335 Ala Val Leu Thr Glu Leu
Ala Phe Ile Asp Asn Gly Ile Asp Tyr Ser 340 345 350 Lys Leu Ser Thr
Glu Asn Gly Arg Gln Ile Ala Ala Glu Ala Ile Tyr 355 360 365 Glu Gly
Ile Leu Asp Tyr Tyr Glu Trp Lys Gly Asn Asn Val Ser Glu 370 375 380
Tyr Arg Leu 385 <210> SEQ ID NO 10 <211> LENGTH: 410
<212> TYPE: PRT <213> ORGANISM: Bacillus anthracis
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: NCBI/NP_844897 <309> DATABASE ENTRY DATE: 2017-10-06
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(410) <400>
SEQUENCE: 10 Met Lys Asn Lys Leu Ile Ala Thr Gly Ile Leu Ala Gly
Ser Leu Leu 1 5 10 15 Ser Tyr Ser Thr Ser Ile Val Ala Asp Thr His
Arg Phe Pro Asp Val 20 25 30 Pro Ala Trp Ala Asp Lys Ser Val Thr
Tyr Leu Val Asp Lys Gln Val 35 40 45 Leu Ser Gly Tyr Pro Asp Gly
Thr Phe Gly Ser Ser Asp Thr Leu Asp 50 55 60 Arg Ala Ser Ala Ala
Thr Ile Met Thr Lys Ala Leu Gly Ile His Ile 65 70 75 80 Asp Leu Asn
Ala Lys Pro Ser Phe Lys Asp Ser Gln Asn His Trp Gly 85 90 95 Thr
Pro Tyr Ile Ala Ala Ala Glu Lys Ala Gly Ile Ile Lys Gly Glu 100 105
110 Gly Asn Gly Ile Phe Asn Pro Ser Gly Lys Val Thr Arg Ala Ala Met
115 120 125 Ala Thr Met Leu Val Asn Ala Tyr Lys Leu Gln Asn Lys Asn
Thr Ser 130 135 140 Asn Gly Gln Ser Lys Phe Glu Asp Leu Lys Gly His
Trp Gly Glu Lys 145 150 155 160 Phe Ala Asn Thr Leu Ile Asp Leu Lys
Ile Ser Val Gly Thr Asp Asn 165 170 175 Gly Trp Gln Pro Asn Lys Phe
Ile Thr Arg Ala Glu Ala Ala Gln Leu 180 185 190 Thr Ala Lys Thr Asp
Met Leu Gln Tyr Ser His Ser Asn Pro Leu Glu 195 200 205 Asn Lys Thr
Ile Ile Ile Asp Pro Gly His Gly Gly Glu Asp Pro Gly 210 215 220 Lys
Asp Thr Lys Gly Leu Pro Glu Ser Lys Ile Val Leu Asp Thr Ser 225 230
235 240 Leu Arg Leu Gln Lys Leu Leu Glu Lys His Thr Pro Phe Thr Val
Leu 245 250 255 Leu Thr Arg Lys Ser Asp Thr Arg Pro Gly His Asp Gln
Lys Ser Ser 260 265 270 Leu Gln Glu Arg Val Lys Phe Ala Lys Gln Asn
Gln Gly Asp Ile Phe 275 280 285 Ile Ser Val His Ala Asn Ala Phe Asn
Gly Asn Ala Lys Gly Thr Glu 290 295 300 Thr Tyr Tyr Tyr Lys Ser Ser
Lys Ser Glu Lys Thr Asn Pro His Val 305 310 315 320 Glu Glu Ser Arg
Val Leu Ala Glu Lys Ile Gln Thr Arg Leu Val Asp 325 330 335 Ala Leu
Gln Thr Arg Asp Arg Gly Val Lys His Gly Asp Leu His Val 340 345 350
Ile Arg Glu Asn Asp Met Pro Ala Val Leu Thr Glu Leu Ala Phe Ile 355
360 365 Asp Asn Gly Ile Asp Tyr Ser Lys Leu Ser Thr Glu Asn Gly Arg
Gln 370 375 380 Ile Ala Ala Glu Ala Ile Tyr Glu Gly Ile Leu Asp Tyr
Tyr Glu Trp 385 390 395 400 Lys Gly Asn Asn Val Ser Glu Tyr Arg Leu
405 410 <210> SEQ ID NO 11 <211> LENGTH: 348
<212> TYPE: PRT <213> ORGANISM: Bacillus
weihenstephanensis <300> PUBLICATION INFORMATION: <308>
DATABASE ACCESSION NUMBER: NCBI/YP_001646166.1 <309> DATABASE
ENTRY DATE: 2014-12-16 <313> RELEVANT RESIDUES IN SEQ ID NO:
(1)..(348) <400> SEQUENCE: 11 Met Gln Asn Arg Ser Ser Ser Asn
Ile Thr Phe Ile Asp Val Ser His 1 5 10 15 Trp Glu Gly Asn Ile Asn
Trp Asn Ala Val Lys Ser Ser Gly Ile Pro 20 25 30 Ala Ala Tyr Ala
Lys Ala Thr Glu Gly Val Asn Tyr Ile Asp Pro Thr 35 40 45 Phe Val
Gln Asn Val Gln Ala Ala Arg Asn Ala Asn Val Leu Ile Gly 50 55 60
Ala Tyr His Phe Ala His Pro Glu Gln Asn Asp Ala Ile Ser Glu Ala 65
70 75 80 Lys Tyr Phe Val Ser Ile Leu Gln Ser Asn Gln Thr Asp Leu
Ile Pro 85 90 95 Val Leu Asp Leu Glu Ser Pro Thr Asp Thr Ser Asn
Ser Ser Leu Thr 100 105 110 Gly Ala Thr Ile Ser Asn Trp Ala Arg Ser
Phe Val Asn Tyr Val Lys 115 120 125 Gln Ala Thr Gly Lys Asp Val Met
Leu Tyr Thr Gly Ile Trp Tyr Ile 130 135 140 Asn Glu Phe Gly Ile Ser
Gly Leu Ser Asp Ile Pro Leu Trp Ile Ser 145 150 155 160 Lys Tyr Ser
Ser Ile Pro Pro Ala Asp Ala Gly Gly Trp Thr Glu Trp 165 170 175 Thr
Ala Trp Gln Tyr Thr Asp Ser Gly Gln Ile Ser Gly Val Gly Asn 180 185
190 Cys Asp Val Ser Ala Ala Val Ser Leu Glu Ala Leu Gln Gly Asn Gly
195 200 205 Ala Ser Gly Gly Gly Asn Val Ser Thr Pro Asn Asn Ala Thr
Pro Val 210 215 220 Tyr Gly Val Ala Val Ile Asn Gly Asp Asn Val Asn
Leu Arg Ser Gly 225 230 235 240 Pro Ser Leu Gln Ser Ser Val Ile Arg
Gln Leu Asn Arg Gly Glu Ser 245 250 255 Tyr Glu Val Trp Gly Glu Gln
Asn Gly Trp Leu Cys Leu Gly Thr Asn 260 265 270 Gln Trp Val Tyr Asn
Asp Ser Ser Tyr Ile Gln Tyr Lys His Tyr Val 275 280 285 Ala Thr Ile
Thr Gly Asp Asn Val Asn Leu Arg Asp Ala Pro Ser Leu 290 295 300 Asn
Gly Asn Val Ile Arg Gln Leu His His Gly Glu Ser Tyr Arg Val
305 310 315 320 Trp Ser Lys Gln Asp Gly Trp Leu Cys Leu Gly Thr Asn
Gln Trp Val 325 330 335 Tyr Tyr Asp Ser Ser Tyr Ile Gln Tyr Gly Val
Gln 340 345 <210> SEQ ID NO 12 <211> LENGTH: 529
<212> TYPE: PRT <213> ORGANISM: Bacillus anthracis
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: NCBI/NP_843409 <309> DATABASE ENTRY DATE: 2017-10-06
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(529) <400>
SEQUENCE: 12 Met Lys Tyr Arg Ala Val Ala Ala Gly Ile Leu Ala Ala
Ser Leu Leu 1 5 10 15 Ser Ser Pro Val Ser Ser Phe Ala Ala Ala Lys
Lys Phe Ser Asp Val 20 25 30 Pro Thr Trp Ala Gln Gln Ser Val Asp
Tyr Leu Val Gly Lys Lys Ala 35 40 45 Leu Asp Gly Lys Pro Asp Gly
Thr Phe Ser Pro Ser Glu Ala Val Asp 50 55 60 Arg Gly Ser Ala Ala
Lys Ile Leu Ala Val Val Leu Gly Leu Pro Val 65 70 75 80 Asp Pro Lys
Ala Lys Pro Ser Phe Lys Asp Ala Gln Asn His Trp Ala 85 90 95 Ala
Pro Tyr Ile Ala Ala Val Glu Lys Ala Gly Val Ile Asn Gly Asp 100 105
110 Gly Thr Gly Lys Phe Asn Pro Ser Ser Gln Ile Asn Arg Ala Ser Met
115 120 125 Ala Ser Met Leu Val Gln Ala Tyr Ser Leu Asp Lys Lys Ile
Ile Gly 130 135 140 Glu Leu Pro Thr Gln Phe Lys Asp Leu Glu Pro His
Trp Gly Lys Lys 145 150 155 160 Gln Ala Asn Ile Leu Val Ala Leu Glu
Ile Ser Lys Gly Thr Gly Asn 165 170 175 Gly Trp Asn Pro Glu Gly Thr
Val Thr Arg Ala Glu Ala Ala Gln Phe 180 185 190 Ile Ala Met Ala Asp
Gln Asn Lys Thr Ser Thr Ser Lys Arg Met Tyr 195 200 205 Met Asn Arg
Asn Val Ile Thr Tyr His Gln Pro Ser Leu Ser Ser Gly 210 215 220 Ile
Thr Asp Val Gln His Lys Pro Gln Met Val Glu Val Thr Glu Gln 225 230
235 240 Arg Ala Asp Gly Trp Leu Lys Ile Val Thr Ser Lys Gly Glu Lys
Trp 245 250 255 Thr Pro Leu Thr Glu Lys Thr Glu Thr Ile Asn Glu Glu
Phe Thr Thr 260 265 270 Tyr Glu Thr Ala Ser His Ser Ser Lys Val Leu
Gly Thr Tyr Asn Ala 275 280 285 Gln Thr Val Thr Val Met Glu Glu Ser
Gly Ser Trp Ile Arg Ile Arg 290 295 300 Val Gly Ala Gly Phe Gln Trp
Val Asp Lys Asn Gln Leu Asn Pro Val 305 310 315 320 Lys Gln Glu Asn
Phe Leu Glu Gly Lys Ala Ile Ile Ile Asp Pro Gly 325 330 335 His Gly
Gly Ile Asp Ser Gly Asn Val Gly Tyr Tyr Glu Lys Glu Ser 340 345 350
Glu Thr Val Leu Asp Val Ser Leu Arg Leu Lys Lys Ile Phe Glu Gln 355
360 365 Lys Ala Pro Phe Thr Val Met Phe Thr Arg Thr Asp Asn Thr Arg
Pro 370 375 380 Gly Val Asn Ser Thr Asp Ser Leu Lys Lys Arg Val Glu
Phe Ala Gln 385 390 395 400 Glu His Asn Gly Asp Ile Phe Val Ser Ile
His Ala Asn Gly Ser Ala 405 410 415 Glu Lys Asn Gly Gln Gly Thr Glu
Thr Leu Tyr Tyr Gln Ser Ala Arg 420 425 430 Ala Lys Val Thr Asn Pro
His Val Glu Asp Ser Lys Leu Leu Ala Gln 435 440 445 Lys Ile Gln Asp
Arg Leu Val Ala Ala Leu Gly Thr Lys Asp Arg Gly 450 455 460 Val Lys
His Gln Asp Leu Tyr Val Thr Arg Glu Asn Thr Met Pro Ala 465 470 475
480 Val Leu Thr Glu Leu Ala Phe Val Asp Asn Lys Ser Asp Ala Asp Lys
485 490 495 Ile Ala Thr Pro Lys Gln Arg Gln Ala Ala Ala Glu Ala Ile
Tyr Gln 500 505 510 Gly Ile Leu Asp Tyr Tyr Glu Ala Lys Gly Asn Asn
Val Ser Ser Phe 515 520 525 Arg
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