U.S. patent application number 12/687267 was filed with the patent office on 2010-05-13 for polypeptides having antimicrobial activity and polynucleotides encoding same.
This patent application is currently assigned to Novozymes Adenium Biotech A/S. Invention is credited to Hans-Henrik Kristensen Hoegenhaug, Per Hose Mygind, Dorotea Raventos Segura, Olivier Taboureau, Jesper Vind.
Application Number | 20100120690 12/687267 |
Document ID | / |
Family ID | 59220460 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100120690 |
Kind Code |
A1 |
Vind; Jesper ; et
al. |
May 13, 2010 |
Polypeptides Having Antimicrobial Activity and Polynucleotides
Encoding Same
Abstract
The present invention relates to isolated polypeptides having
antimicrobial activity and isolated polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods for producing and using the polypeptides.
Inventors: |
Vind; Jesper; (Vaerlose,
DK) ; Segura; Dorotea Raventos; (Humlebaek, DK)
; Hoegenhaug; Hans-Henrik Kristensen; (Holte, DK)
; Mygind; Per Hose; (Soeborg, DK) ; Taboureau;
Olivier; (Kgs. Lyngby, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes Adenium Biotech
A/S
Bagsvaerd
DK
|
Family ID: |
59220460 |
Appl. No.: |
12/687267 |
Filed: |
January 14, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11446896 |
Jun 5, 2006 |
7671175 |
|
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12687267 |
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60726992 |
Oct 14, 2005 |
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60688589 |
Jun 8, 2005 |
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Current U.S.
Class: |
514/1.1 ;
426/335; 435/254.21; 435/320.1; 510/477; 530/324; 536/23.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 31/04 20180101; A23K 20/147 20160501; A23L 33/18 20160801;
A61P 31/06 20180101; C07K 14/37 20130101 |
Class at
Publication: |
514/12 ; 530/324;
536/23.1; 435/320.1; 435/254.21; 510/477; 426/335 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; A61P 31/04 20060101
A61P031/04; A61P 31/06 20060101 A61P031/06; C07H 21/00 20060101
C07H021/00; C12N 15/74 20060101 C12N015/74; C12N 1/19 20060101
C12N001/19; C11D 3/30 20060101 C11D003/30; A23L 3/34 20060101
A23L003/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 6, 2005 |
DK |
2005 00823 |
Oct 13, 2005 |
DK |
2005 01435 |
Claims
1. A polypeptide having antimicrobial activity, which comprises an
amino acid sequence which has at least 80% identity with amino
acids 1 to 40 of SEQ ID NO: 2: TABLE-US-00010
G-X.sub.1-G-C-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.su-
b.9-X.sub.10-X.sub.11-C-H-X.sub.12-
X.sub.13-C-X.sub.14-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20--
G-G-X.sub.21-C-X.sub.22-
X.sub.23-X.sub.24-X.sub.25-X.sub.26-X.sub.27-C-X.sub.28-C-X.sub.29;
wherein X.sub.1=F, L, W or I; preferably X.sub.1=F; X.sub.2=N, R,
Q, V, G, S, A, K, L, M, D, H or Y; preferably X.sub.2=N, R, Q, V,
G, S, A, K or Y; X.sub.3=G, R, A or K; preferably X.sub.3=G;
X.sub.4=P, A, L, V, K or R; preferably X.sub.4=P, K or R; X.sub.5=W
or R; X.sub.6=D, A, G, K, L, T, N, F, H, M, P, Q, S, C, I, R, V or
Y; preferably X.sub.6=D, A, G, K, L, T, N, F, H, M, P, Q, S, V or
Y; X.sub.7=E, G, A, L, C, Q or S; preferably X.sub.7=E, G or S;
X.sub.8=D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M, A, C or R;
preferably X.sub.8=D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or
R; X.sub.9=D or P; preferably X.sub.9=D; X.sub.10=M, R, S, V, A, F,
G, L, T, Y, W, E or K; preferably X.sub.10=M, R, S, V, G, Y, L, F,
T, W or K; X.sub.11=Q, R, L, F, G, H, S, A, C, I, K, M, P, T, V, W
or Y; preferably X.sub.11=Q, R, L, F, G, H, S, K or Y; X.sub.12=N,
R, I, Y, V, K, T, Q, S, A, W, E or H; X.sub.13=H, A, F, Q, T, V or
L; preferably X.sub.13=H or L; X.sub.14=K, Q or R; preferably
X.sub.14=K or R; X.sub.15=S, A, V, N or F; X.sub.16=I, L, M, T, W
or V; preferably X.sub.16=I, L or V; X.sub.17=K, T or R;
X.sub.18=G, H, K, A, P, F, I, Q, R, S, T, Y or N; preferably
X.sub.18=G, H, R, K or N; X.sub.19=Y, H, K, L, M, N, Q, S, V or R;
preferably X.sub.19=Y or R; X.sub.20=K, F, H, T, C or R; preferably
X.sub.20=K or R; X.sub.21=Y, F, R, A, H, L, M, S or W; preferably
X.sub.21=Y, F, R or W; X.sub.22=A, K, N, Q, T, E, H, I, R, S, V, G
or Y; preferably X.sub.22=A, K, N, Q, T, S or Y; X.sub.23=K, R or
T; preferably X.sub.23=K or R; X.sub.24=G, K, Q, E, N, S, T, A or
R; preferably X.sub.24=G, K, Q, A or R; X.sub.25=G, K, H, W or R;
preferably X.sub.25=G, K or R; X.sub.26=F, A, H, I, M, V, W, R or
L; preferably X.sub.26=F or L; X.sub.27=V, L, M, I, K, Q, R or T;
preferably X.sub.27=V, L, M or T; X.sub.28=K, H, N or R; preferably
X.sub.28=K or R; X.sub.29=Y, I, YRCG or YR; preferably X.sub.29=Y
or YR; and which has less than 100% identity with amino acids 1 to
40 of SEQ ID NO: 1.
2. The polypeptide of claim 1, wherein the amino acid sequence has
at least 85% identity with amino acids 1 to 40 of SEQ ID NO: 2,
preferably at least 90% identity, or at least 95% identity with
amino acids 1 to 40 of SEQ ID NO: 2.
3. The polypeptide of claim 1, which comprises the amino acid
sequence of SEQ ID NO: 2.
4. The polypeptide of claim 1, which comprises the amino acid
sequence of anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of
SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ
ID NO: 274.
5. The polypeptide of claim 1, which consists of the amino acid
sequence of SEQ ID NO: 2; or a fragment thereof having
antimicrobial activity and comprising 6 cysteine residues.
6. The polypeptide of claim 5, which consists of the amino acid
sequence of anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of
SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ
ID NO: 274; or a fragment thereof having antimicrobial activity and
comprising 6 cysteine residues.
7. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
8. The composition of claim 7, which further comprises an
additional biocidal agent.
9. A detergent composition comprising a surfactant and the
polypeptide of claim 1.
10. An animal feed additive comprising (a) a polypeptide of claim
1; and (b) a fat soluble vitamin, and/or (c) a water soluble
vitamin, and/or (d) a trace mineral, and/or (e) a macro
mineral.
11. The animal feed additive of claim 10, which further comprises
phytase, xylanase, galactanase, and/or beta-glucanase.
12. An animal feed composition having a crude protein content of 50
to 800 g/kg and comprising a polypeptide of claim 1.
13. A method for killing or inhibiting growth of microbial cells
comprising contacting the microbial cells with a polypeptide of
claim 1.
14. A method of treating a microbial infection, comprising
administering to a human or animal a polypeptide of claim 1 in an
amount effective to treat the microbial infection.
15. A method of treating tuberculosis, comprising administering to
a human or animal a polypeptide of claim 1 in an amount effective
to treat the tuberculosis.
16. A polynucleotide comprising a nucleotide sequence which encodes
a polypeptide of any of claim 1.
17. A nucleic acid construct comprising the polynucleotide of claim
16 operably linked to one or more control sequences that direct the
production of the polypeptide in an expression host.
18. A recombinant expression vector comprising the nucleic acid
construct of claim 17.
19. A recombinant host cell comprising the nucleic acid construct
of claim 17.
20. A method for producing a polypeptide having antimicrobial
activity, comprising (a) cultivating the recombinant host cell of
claim 19 under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/446,896 filed Jun. 5, 2006, which claims priority or the
benefit under 35 U.S.C. 119 of Danish application nos. PA 2005
00823 and PA 2005 01435 filed Jun. 6, 2005 and Oct. 13, 2005,
respectively, and U.S. provisional application Nos. 60/726,992 and
60/688,589 filed Oct. 14, 2005 and June 8, respectively, the
contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to isolated polypeptides
having antimicrobial activity and isolated polynucleotides encoding
the polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods for producing and using the polypeptides.
BACKGROUND OF THE INVENTION
[0003] It is an object of the present invention to provide
polypeptides having improved antimicrobial activity. The
polypeptides may exhibit reduced hemolytic activity and/or reduced
cytotoxicity. The polypeptides may also exhibit reduced sensitivity
towards cations, such as Ca.sup.2+, Mg.sup.2+, Na.sup.+. The
polypeptides may also exhibit a different antimicrobial spectrum
compared to SEQ ID NO: 1.
SUMMARY OF THE INVENTION
[0004] The present invention provides a polypeptide having
antimicrobial activity which comprises, preferably consists of, an
amino acid sequence which has at least 80% identity with amino
acids 1 to 40 of the amino acid sequence:
TABLE-US-00001
G-X.sub.1-G-C-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-C-H-X.sub.12-
X.sub.13-C-X.sub.14-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20--
G-G-X.sub.21-C-X.sub.22-
X.sub.23-X.sub.24-X.sub.25-X.sub.26-X.sub.27-C-X.sub.28-C-X.sub.29;
wherein X.sub.1=F, L, W or I; preferably X.sub.1=F; X.sub.2=N, R,
Q, V, G, S, A, K, L, M, D, H or Y; preferably X.sub.2=N, R, Q, V,
G, S, A, K or Y; X.sub.3=G, R, A or K; preferably X.sub.3=G;
X.sub.4=P, A, L, V, K or R; preferably X.sub.4=P, K or R;
X.sub.5=W or R;
[0005] X.sub.6=D, A, G, K, L, T, N, F, H, M, P, Q, S, C, I, R, V or
Y; preferably X.sub.6=D, A, G, K, L, T, N, F, H, M, P, Q, S, V or
Y; X.sub.7=E, G, A, L, C, Q or S; preferably X.sub.7=E, G or S;
X.sub.8=D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M, A, C or R;
preferably X.sub.8=D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or
R; X.sub.9=D or P; preferably X.sub.9=D; X.sub.10=M, R, S, V, A, F,
G, L, T, Y, W, E or K; preferably X.sub.10=M, R, S, V, G, Y, L, F,
T, W or K; X.sub.11=Q, R, L, F, G, H, S, A, C, I, K, M, P, T, V, W
or Y; preferably X.sub.11=Q, R, L, F, G, H, S, K or Y;
X.sub.12=N, R, I, Y, V, K, T, Q, S, F, A, W, E or H;
[0006] X.sub.13=H, A, F, Q, T, V or L; preferably X.sub.13=H or L;
X.sub.14=K, Q or R; preferably X.sub.14=K or R;
X.sub.15=S, A, V, N or F;
[0007] X.sub.16=I, L, M, T, W or V; preferably X.sub.16=I, L or
V;
X.sub.17=K, T or R;
[0008] X.sub.18=G, H, K, A, P, F, I, Q, R, S, T, Y or N; preferably
X.sub.18=G, H, R, K or N; X.sub.19=Y, H, K, L, M, N, Q, S, V or R;
preferably X.sub.19=Y or R; X.sub.20=K, F, H, T, C or R; preferably
X.sub.20=K or R; X.sub.21=Y, F, R, A, H, L, M, S or W; preferably
X.sub.21=Y, F, R or W; X.sub.22=A, K, N, Q, T, E, H, I, R, S, V, G
or Y; preferably X.sub.22=A, K, N, Q, T, S or Y; X.sub.23=K, R or
T; preferably X.sub.23=K or R; X.sub.24=G, K, Q, E, N, S, T, A or
R; preferably X.sub.24=G, K, Q, A or R; X.sub.25=G, K, H, W or R;
preferably X.sub.25=G, K or R; X.sub.26=F, A, H, I, M, V, W, R or
L; preferably X.sub.26=F or L; X.sub.27=V, L, M, I, K, Q, R or T;
preferably X.sub.27=V, L, M or T; X.sub.28=K, H, N or R; preferably
X.sub.28=K or R; X.sub.29=Y, I, YRCG or YR; preferably X.sub.29=Y
or YR; and which has less than 100% identity with amino acids 1 to
40 of SEQ ID NO: 1.
[0009] The present invention also relates to nucleic acid
constructs, recombinant expression vectors, and recombinant host
cells comprising the polynucleotides.
[0010] The present invention also relates to methods for producing
such polypeptides having antimicrobial activity comprising (a)
cultivating a recombinant host cell comprising a nucleic acid
construct comprising a polynucleotide encoding the polypeptide
under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.
[0011] The present invention also relates to methods of using the
polypeptides of the invention.
DEFINITIONS
[0012] Antimicrobial activity: The term "antimicrobial activity" is
defined herein as an activity which is capable of killing or
inhibiting growth of microbial cells. In the context of the present
invention the term "antimicrobial" is intended to mean that there
is a bactericidal and/or a bacteriostatic and/or fungicidal and/or
fungistatic effect and/or a virucidal effect, wherein the term
"bactericidal" is to be understood as capable of killing bacterial
cells. The term "bacteriostatic" is to be understood as capable of
inhibiting bacterial growth, i.e., inhibiting growing bacterial
cells. The term "fungicidal" is to be understood as capable of
killing fungal cells. The term "fungistatic" is to be understood as
capable of inhibiting fungal growth, i.e., inhibiting growing
fungal cells. The term "virucidal" is to be understood as capable
of inactivating virus. The term "microbial cells" denotes bacterial
or fungal cells (including yeasts).
[0013] In the context of the present invention the term "inhibiting
growth of microbial cells" is intended to mean that the cells are
in the non-growing state, i.e., that they are not able to
propagate.
[0014] For purposes of the present invention, antimicrobial
activity may be determined according to the procedure described by
Lehrer et al., 1991, Journal of Immunological Methods 137(2):
167-174. Alternatively, antimicrobial activity may be determined
according to the NCCLS guidelines from CLSI (Clinical and
Laboratory Standards Institute; formerly known as National
Committee for Clinical and Laboratory Standards).
[0015] Polypeptides having antimicrobial activity may be capable of
reducing the number of living cells of Escherichia coli (DSM 1576)
to 1/100 after 24 hours (preferably after 12 hours, more preferably
after 8 hours, more preferably after 4 hours, more preferably after
2 hours, most preferably after 1 hour, and in particular after 30
minutes) incubation at 20.degree. C. in an aqueous solution of 25%
(w/w); preferably in an aqueous solution of 10% (w/w); more
preferably in an aqueous solution of 5% (w/w); even more preferably
in an aqueous solution of 1% (w/w); most preferably in an aqueous
solution of 0.5% (w/w); and in particular in an aqueous solution of
0.1% (w/w) of the polypeptides having antimicrobial activity.
[0016] Polypeptides having antimicrobial activity may also be
capable of inhibiting the outgrowth of Escherichia coli (DSM 1576)
for 24 hours at 25.degree. C. in a microbial growth substrate, when
added in a concentration of 1000 ppm; preferably when added in a
concentration of 500 ppm; more preferably when added in a
concentration of 250 ppm; even more preferably when added in a
concentration of 100 ppm; most preferably when added in a
concentration of 50 ppm; and in particular when added in a
concentration of 25 ppm.
[0017] Polypeptides having antimicrobial activity may be capable of
reducing the number of living cells of Bacillus subtilis (ATCC
6633) to 1/100 after 24 hours (preferably after 12 hours, more
preferably after 8 hours, more preferably after 4 hours, more
preferably after 2 hours, most preferably after 1 hour, and in
particular after 30 minutes) incubation at 20.degree. C. in an
aqueous solution of 25% (w/w); preferably in an aqueous solution of
10% (w/w); more preferably in an aqueous solution of 5% (w/w); even
more preferably in an aqueous solution of 1% (w/w); most preferably
in an aqueous solution of 0.5% (w/w); and in particular in an
aqueous solution of 0.1% (w/w) of the polypeptides having
antimicrobial activity.
[0018] Polypeptides having antimicrobial activity may also be
capable of inhibiting the outgrowth of Bacillus subtilis (ATCC
6633) for 24 hours at 25.degree. C. in a microbial growth
substrate, when added in a concentration of 1000 ppm; preferably
when added in a concentration of 500 ppm; more preferably when
added in a concentration of 250 ppm; even more preferably when
added in a concentration of 100 ppm; most preferably when added in
a concentration of 50 ppm; and in particular when added in a
concentration of 25 ppm.
[0019] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100% of
the antimicrobial activity of the polypeptide consisting of the
amino acid sequence shown as amino acids 1 to 40 of anyone of SEQ
ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID
NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274.
[0020] Defensin: The term "defensin" as used herein refers to
polypeptides recognized by a person skilled in the art as belonging
to the defensin class of antimicrobial peptides. To determine if a
polypeptide is a defensin according to the invention, the amino
acid sequence is preferably compared with the hidden markov model
profiles (HMM profiles) of the PFAM database by using the freely
available HMMER software package (see Example 7).
[0021] The PFAM defensin families include Defensin.sub.--1 or
"Mammalian defensin" (accession no. PF00323), Defensin.sub.--2 or
"Arthropod defensin" (accession no. PF01097), Defensin_beta or
"Beta Defensin" (accession no. PF00711), Defensin_propep or
"Defensin propeptide" (accession no. PF00879) and Gamma-thionin or
"Gamma-thionins family" (accession no. PF00304).
[0022] The defensins may belong to the alpha-defensin class, the
beta-defensin class, the theta-defensin class, the insect or
arthropod defensin classes, or the plant defensin class.
[0023] In an embodiment, the amino acid sequence of a defensin
according to the invention comprises 4, 5, 6, 7, or 8 cysteine
residues, preferably 4, 5, or 6 cysteine residues, more preferably
4 or 6 cysteine residues, and most preferably 6 cysteine
residues.
[0024] The defensins may also be synthetic defensins sharing the
characteristic features of any of the defensin classes.
[0025] Examples of such defensins include, but are not limited to,
.alpha.-Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3;
.beta.-Defensin-12, Drosomycin, Heliomicin, .gamma.1-purothionin,
Insect defensin A, and the defensins disclosed in PCT applications
WO 99/53053, WO 02/06324, WO 02/085934, PCT/DK2005/000725,
PCT/DK2005/000735 and PCT/DK2006/000155.
[0026] Isolated polypeptide: The term "isolated polypeptide" as
used herein refers to a polypeptide which is at least 20% pure,
preferably at least 40% pure, more preferably at least 60% pure,
even more preferably at least 80% pure, most preferably at least
90% pure, and even most preferably at least 95% pure, as determined
by SDS-PAGE.
[0027] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation which
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%, at
most 3%, even more preferably at most 2%, most preferably at most
1%, and even most preferably at most 0.5% by weight of other
polypeptide material with which it is natively associated. It is,
therefore, preferred that the substantially pure polypeptide is at
least 92% pure, preferably at least 94% pure, more preferably at
least 95% pure, more preferably at least 96% pure, more preferably
at least 96% pure, more preferably at least 97% pure, more
preferably at least 98% pure, even more preferably at least 99%,
most preferably at least 99.5% pure, and even most preferably 100%
pure by weight of the total polypeptide material present in the
preparation.
[0028] The polypeptides of the present invention are preferably in
a substantially pure form. In particular, it is preferred that the
polypeptides are in "essentially pure form", i.e., that the
polypeptide preparation is essentially free of other polypeptide
material with which it is natively associated. This can be
accomplished, for example, by preparing the polypeptide by means of
well-known recombinant methods or by classical purification
methods.
[0029] Herein, the term "substantially pure polypeptide" is
synonymous with the terms "isolated polypeptide" and "polypeptide
in isolated form."
[0030] Variant: The term "variant" is defined herein as an
antimicrobial polypeptide comprising one or more alterations, such
as substitutions, insertions, deletions, and/or truncations of one
or more specific amino acid residues at one or more specific
positions in the polypeptide.
[0031] Numbering of Variants: In the present invention, a specific
numbering of amino acid residue positions in the antimicrobial
polypeptide variants is employed. For example, by aligning the
amino acid sequences of known antimicrobial polypeptides, it is
possible to designate an amino acid position number to any amino
acid residue in any antimicrobial polypeptide.
[0032] Using the numbering system originating from the amino acid
sequence of the antimicrobial polypeptide disclosed in SEQ ID NO:
1, aligned with the amino acid sequence of a number of other
antimicrobial polypeptides, it is possible to indicate the position
of an amino acid residue in an antimicrobial polypeptide in regions
of structural homology.
[0033] Multiple alignments of protein sequences may be made, for
example, using "ClustalW" (Thompson, Higgins, and Gibson, 1994,
CLUSTAL W: Improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, positions-specific
gap penalties and weight matrix choice, Nucleic Acids Research 22:
4673-4680). Multiple alignments of DNA sequences may be done using
the protein alignment as a template, replacing the amino acids with
the corresponding codon from the DNA sequence.
[0034] Pairwise sequence comparison algorithms in common use are
adequate to detect similarities between protein sequences that have
not diverged beyond the point of approximately 20-30% sequence
identity (Doolittle, 1992, Protein Sci. 1: 191-200; Brenner et al.,
1998, Proc. Natl. Acad. Sci. USA 95, 6073-6078). However, truly
homologous proteins with the same fold and similar biological
function have often diverged to the point where traditional
sequence-based comparison fails to detect their relationship
(Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Greater
sensitivity in sequence-based searching can be attained using
search programs that utilize probabilistic representations of
protein families (profiles) to search databases. For example, the
PSI-BLAST program generates profiles through an iterative database
search process and is capable of detecting remote homologs (Atschul
et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater
sensitivity can be achieved if the family or superfamily for the
protein of interest has one or more representatives in the protein
structure databases. Programs such as GenTHREADER (Jones, 1999, J.
Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics
19: 874-881) utilize information from a variety of sources
(PSI-BLAST, secondary structure prediction, structural alignment
profiles, and solvation potentials) as input to a neural network
that predicts the structural fold for a query sequence. Similarly,
the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can
be used to align a sequence of unknown structure with the
superfamily models present in the SCOP database. These alignments
can in turn be used to generate homology models for the protein of
interest, and such models can be assessed for accuracy using a
variety of tools developed for that purpose.
[0035] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
For example the SCOP superfamilies of proteins have been
structurally aligned, and those alignments are accessible and
downloadable. These alignments can be used to predict the
structurally and functionally corresponding amino acid residues in
proteins within the same structural superfamily. This information,
along with information derived from homology modeling and profile
searches, can be used to predict which residues to mutate when
moving mutations of interest from one protein to a close or remote
homolog.
[0036] In describing the various antimicrobial polypeptide variants
of the present invention, the nomenclature described below is
adapted for ease of reference. In all cases, the accepted IUPAC
single letter or triple letter amino acid abbreviation is
employed.
[0037] For an amino acid substitution, the following nomenclature
is used: Original amino acid, position, substituted amino acid(s).
Accordingly, the substitution of threonine with alanine at position
226 is designated as "T226A"; and substitution of tyrosine with
tyrosine and arginine at position 40 (effectively adding arginine
after tyrosine) is designated as "Y40YR". Multiple mutations are
separated by addition marks ("+"), e.g., "G205R+S411F",
representing mutations at positions 205 and 411 substituting
glycine (G) with arginine (R), and serine (S) with phenylalanine
(F), respectively.
[0038] Parent antimicrobial polypeptide: The term "parent"
antimicrobial polypeptide as used herein means an antimicrobial
polypeptide to which modifications, e.g., substitution(s),
insertion(s), deletion(s), and/or truncation(s), are made to
produce the antimicrobial polypeptide variants of the present
invention. This term also refers to the polypeptide with which a
variant is compared and aligned. The parent may be a naturally
occurring (wild type) polypeptide, or it may even be a variant
thereof, prepared by any suitable means. For instance, the parent
protein may be a variant of a naturally occurring polypeptide which
has been modified or altered in the amino acid sequence. A parent
may also be an allelic variant which is a polypeptide encoded by
any of two or more alternative forms of a gene occupying the same
chromosomal locus.
[0039] Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"identity".
[0040] For purposes of the present invention, the degree of
identity between two amino acid sequences is determined by using
the program FASTA included in version 2.0.times. of the FASTA
program package (see Pearson and Lipman, 1988, "Improved Tools for
Biological Sequence Analysis", PNAS 85: 2444-2448; and Pearson,
1990, "Rapid and Sensitive Sequence Comparison with FASTP and
FASTA", Methods in Enzymology 183: 63-98). The scoring matrix used
was BLOSUM50, gap penalty was -12, and gap extension penalty was
-2.
[0041] The degree of identity between two nucleotide sequences is
determined using the same algorithm and software package as
described above. The scoring matrix used was the identity matrix,
gap penalty was -16, and gap extension penalty was -4.
[0042] Alternatively, an alignment of two amino acid sequences is
determined by using the Needle program from the EMBOSS package
(http://emboss.org) version 2.8.0. The Needle program implements
the global alignment algorithm described in Needleman and Wunsch,
1970, J. Mol. Biol. 48: 443-453. The substitution matrix used is
BLOSUM62, gap opening penalty is 10, and gap extension penalty is
0.5. The degree of identity between an amino acid sequence of the
present invention (such as amino acids 1 to 40 of SEQ ID NO: 1) and
a different amino acid sequence is calculated as the number of
exact matches in an alignment of the two sequences, divided by the
length (number of amino acid residues) of the sequence of the
present invention; or alternatively the output of Needle labeled
"longest identity" is used as the percent identity and is
calculated as follows: (Identical Residues.times.100)/(Length of
Alignment Number of Gaps in Alignment). The result is expressed in
percent identity.
[0043] Polypeptide Fragment: The term "polypeptide fragment" is
defined herein as a polypeptide having one or more amino acids
deleted from the amino and/or carboxyl terminus of SEQ ID NO: 2 or
a homologous sequence thereof, wherein the fragment has
antimicrobial activity.
[0044] Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more nucleotides deleted from the
5' and/or 3' end of SEQ ID NO: 1 or a homologous sequence thereof,
wherein the subsequence encodes a polypeptide fragment having
antimicrobial activity.
[0045] Allelic variant: The term "allelic variant" denotes herein
any of two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in polymorphism within populations. Gene
mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequences. An
allelic variant of a polypeptide is a polypeptide encoded by an
allelic variant of a gene.
[0046] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation free of other extraneous or unwanted nucleotides and in
a form suitable for use within genetically engineered protein
production systems. Thus, a substantially pure polynucleotide
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%,
more preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polynucleotide material with which it is natively
associated. A substantially pure polynucleotide may, however,
include naturally occurring 5' and 3' untranslated regions, such as
promoters and terminators. It is preferred that the substantially
pure polynucleotide is at least 90% pure, preferably at least 92%
pure, more preferably at least 94% pure, more preferably at least
95% pure, more preferably at least 96% pure, more preferably at
least 97% pure, even more preferably at least 98% pure, most
preferably at least 99%, and even most preferably at least 99.5%
pure by weight. The polynucleotides of the present invention are
preferably in a substantially pure form. In particular, it is
preferred that the polynucleotides disclosed herein are in
"essentially pure form", i.e., that the polynucleotide preparation
is essentially free of other polynucleotide material with which it
is natively associated. Herein, the term "substantially pure
polynucleotide" is synonymous with the terms "isolated
polynucleotide" and "polynucleotide in isolated form." The
polynucleotides may be of genomic, cDNA, RNA, semisynthetic,
synthetic origin, or any combinations thereof.
[0047] cDNA: The term "cDNA" is defined herein as a DNA molecule
which can be prepared by reverse transcription from a mature,
spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks
intron sequences that are usually present in the corresponding
genomic DNA. The initial, primary RNA transcript is a precursor to
mRNA which is processed through a series of steps before appearing
as mature spliced mRNA. These steps include the removal of intron
sequences by a process called splicing. cDNA derived from mRNA
lacks, therefore, any intron sequences.
[0048] Nucleic acid construct: The term "nucleic acid construct" as
used herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene
or which is modified to contain segments of nucleic acids in a
manner that would not otherwise exist in nature. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence of the present
invention.
[0049] Control sequence: The term "control sequences" is defined
herein to include all components, which are necessary or
advantageous for the expression of a polynucleotide encoding a
polypeptide of the present invention. Each control sequence may be
native or foreign to the nucleotide sequence encoding the
polypeptide. Such control sequences include, but are not limited
to, a leader, polyadenylation sequence, propeptide sequence,
promoter, signal peptide sequence, and transcription terminator. At
a minimum, the control sequences include a promoter, and
transcriptional and translational stop signals. The control
sequences may be provided with linkers for the purpose of
introducing specific restriction sites facilitating ligation of the
control sequences with the coding region of the nucleotide sequence
encoding a polypeptide.
[0050] Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0051] Coding sequence: When used herein the term "coding sequence"
means a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG. The coding sequence may a DNA, cDNA, or
recombinant nucleotide sequence.
[0052] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0053] Expression vector: The term "expression vector" is defined
herein as a linear or circular DNA molecule that comprises a
polynucleotide encoding a polypeptide of the invention, and which
is operably linked to additional nucleotides that provide for its
expression.
[0054] Host cell: The term "host cell", as used herein, includes
any cell type which is susceptible to transformation, transfection,
transduction, and the like with a nucleic acid construct comprising
a polynucleotide of the present invention.
[0055] Modification: The term "modification" means herein any
chemical modification of the polypeptide consisting of amino acids
1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of
SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ
ID NO: 274 as well as genetic manipulation of the DNA encoding that
polypeptide. The modification(s) can be substitution(s),
deletion(s) and/or insertions(s) of the amino acid(s) as well as
replacement(s) of amino acid side chain(s); or use of unnatural
amino acids with similar characteristics in the amino acid
sequence. In particular the modification(s) can be amidations, such
as amidation of the C-terminus.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Antimicrobial Activity
[0056] In a first aspect, the present invention provides a
polypeptide having antimicrobial activity which comprises,
preferably consists of, an amino acid sequence which has at least
70% identity (preferably at least 80% identity, more preferably at
least 85% identity, even more preferably at least 90% identity,
even more preferably at least 95% identity, most preferably 97%
identity, and in particular 100% identity) with amino acids 1 to 40
of the amino acid sequence (I):
TABLE-US-00002
G-X.sub.1-G-C-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.s-
ub.9-X.sub.10-X.sub.11-C-H-X.sub.12-
X.sub.13-C-X.sub.14-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20--
G-G-X.sub.21-C-X.sub.22-
X.sub.23-X.sub.24-X.sub.25-X.sub.26-X.sub.27-C-X.sub.28-C-X.sub.29;
wherein X.sub.1=F, L, W or I; preferably X.sub.1=F; X.sub.2=N, R,
Q, V, G, S, A, K, L, M, D, H or Y; preferably X.sub.2=N, R, Q, V,
G, S, A, K or Y; more preferably X.sub.2=N, R, S or G; X.sub.3=G,
R, A or K; preferably X.sub.3=G; X.sub.4=P, A, L, V, K or R;
preferably X.sub.4=P, K or R;
X.sub.5=W or R;
[0057] X.sub.6=D, A, G, K, L, T, N, F, H, M, P, Q, S, C, I, R, V or
Y; preferably X.sub.6=D, A, G, K, L, T, N, F, H, M, P, Q, S, V or
Y; more preferably X.sub.6=D, S, A, G or N; X.sub.7=E, G, A, L, C,
Q or S; preferably X.sub.7=E, G or S; X.sub.8=D, F, G, N, V, Y, H,
K, L, P, S, T, W, I, M, A, C or R; preferably X.sub.8=D, F, G, N,
V, Y, H, K, L, P, S, T, W, I, M or R; more preferably X.sub.8=D, G
or N; X.sub.9=D or P; preferably X.sub.9=D; X.sub.10=M, R, S, V, A,
F, G, L, T, Y, W, E or K; preferably X.sub.10=M, R, S, V, G, Y, L,
F, T, W or K; more preferably X.sub.10=M, L, G or V; X.sub.11=Q, R,
L, F, G, H, S, A, C, I, K, M, P, T, V, W or Y; preferably
X.sub.11=Q, R, L, F, G, H, S, K or Y; more preferably X.sub.11=Q,
K, R or F; X.sub.12=N, R, I, Y, V, K, T, Q, S, F, A, W, E or H;
more preferably X.sub.12=N, R, V or Q; X.sub.13=H, A, F, Q, T, V or
L; preferably X.sub.13=H or L; X.sub.14=K, Q or R; preferably
X.sub.14=K or R;
X.sub.15=S, A, V, N or F;
[0058] X.sub.16=I, L, M, T, W or V; preferably X.sub.16=I, L or
V;
X.sub.17=K, T or R;
[0059] X.sub.18=G, H, K, A, P, F, I, Q, R, S, T, Y or N; preferably
X.sub.18=G, H, R, K or N; more preferably
X.sub.18=G or R;
[0060] X.sub.19=Y, H, K, L, M, N, Q, S, V or R; preferably
X.sub.19=Y or R; X.sub.20=K, F, H, T, C or R; preferably X.sub.20=K
or R; X.sub.21=Y, F, R, A, H, L, M, S or W; preferably X.sub.21=Y,
F, R or W; X.sub.22=A, K, N, Q, T, E, H, I, R, S, V, G or Y;
preferably X.sub.22=A, K, N, Q, T, S or Y; more preferably
X.sub.22=A, S or T; X.sub.23=K, R or T; preferably X.sub.23=K or R;
X.sub.24=G, K, Q, E, N, S, T, A or R; preferably X.sub.24=G, K, Q,
A or R; more preferably X.sub.24=G or A; X.sub.25=G, K, H, W or R;
preferably X.sub.25=G, K or R; X.sub.26=F, A, H, I, M, V, W, R or
L; preferably X.sub.26=F or L; X.sub.27=V, L, M, I, K, Q, R or T;
preferably X.sub.27=V, L, M or T; more preferably X.sub.27=V or L;
X.sub.28=K, H, N or R; preferably X.sub.28=K or R; X.sub.29=Y, I,
YRCG or YR; preferably X.sub.29=Y or YR; and which has less than
100% identity with amino acids 1 to 40 of SEQ ID NO: 1.
[0061] In an embodiment, the polypeptide of the invention is a
polypeptide having antimicrobial activity which comprises,
preferably consists of, an amino acid sequence which has at least
70% identity (preferably at least 80% identity, more preferably at
least 85% identity, even more preferably at least 90% identity,
even more preferably at least 95% identity, most preferably 97%
identity, and in particular 100% identity) with amino acids 1 to 40
of the amino acid sequence (II):
TABLE-US-00003
G-F-G-C-X.sub.1-G-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub-
.8-C-H-X.sub.9-X.sub.10-C-
X.sub.11-S-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-G-G-X.sub.17-C-X.s-
ub.18-K-X.sub.19-X.sub.20- X.sub.21-X.sub.22-C-K-C-X.sub.23;
wherein X.sub.1=N, R, Q, V, G, S, A, K or Y; preferably X.sub.i=N,
R, S or G;
X.sub.2=P, K or R;
X.sub.3=W or R;
[0062] X.sub.4=D, A, G, K, L, T, N, F, H, M, P, Q, S, V or Y;
preferably X.sub.4=D, S, A, G or N;
X.sub.5=E, G or S;
[0063] X.sub.6=D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or R;
preferably X.sub.6=D, G or N; X.sub.7=M, R, S, V, G, Y, L, F, T, W
or K; preferably X.sub.7=M, L, G or V; X.sub.8=Q, R, L, F, G, H, S,
K or Y; preferably X.sub.8=Q, K, R or F; X.sub.9=N, R, I, Y, V, K,
T, S, Q or H; preferably X.sub.9=N, R, V or Q;
X.sub.10=H or L;
X.sub.11=K or R;
X.sub.12=I, L or V;
X.sub.13=K or R;
[0064] X.sub.14=G, H, R, K or N; preferably X.sub.14=G or R;
X.sub.15=Y or R;
X.sub.16=K or R;
X.sub.17=Y, F, R or W;
[0065] X.sub.18=A, K, N, Q, T, S or Y; preferably X.sub.18=A, S or
T; X.sub.19=G, K, Q, A or R; preferably X.sub.19=G or A;
X.sub.20=G, K or R;
X.sub.21=F or L;
[0066] X.sub.22=V, L, M or T; preferably X.sub.22=V or L;
X.sub.23=Y or YR;
[0067] and which has less than 100% identity with amino acids 1 to
40 of SEQ ID NO: 1.
[0068] In another embodiment, the amino acid sequence (I) and/or
(II) has 1, 2, 3, 4, 5, 6, 7 or 8 amino acid differences compared
to the amino acid sequence of SEQ ID NO: 1. Preferably 1, 2, 3, 4,
5 or 6; more preferably 1, 2, 3, 4 or 5; even more preferably 1, 2,
3 or 4; even more preferably 1, 2 or 3; and most preferably 1 or 2
amino acids are different compared to the amino acid sequence of
SEQ ID NO: 1.
[0069] In another embodiment, the amino acid sequence (I) and/or
(II) has at least 60% identity with amino acids 1 to 40 of SEQ ID
NO: 1, preferably at least 65% identity, at least 70% identity, at
least 75% identity, at least 80% identity, at least 85% identity,
at least 90% identity, or at least 95% identity with amino acids 1
to 40 of SEQ ID NO: 1.
[0070] In another embodiment, the amino acid sequence (I) and/or
(II) has 0, 1, 2, 3, 4 or 5 insertions, preferably 0, 1, 2 or 3
insertions, more preferably 0, 1 or 2 insertions; and 0, 1, 2, 3, 4
or 5 deletions, preferably 0, 1, 2 or 3 deletions, more preferably
0, 1 or 2 deletions, as compared to SEQ ID NO: 1 or anyone of SEQ
ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID
NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274.
[0071] In another embodiment, the polypeptide of the invention
comprises, preferably consists of, an amino acid sequence which has
at least 60% identity (preferably 70% identity, more preferably 80%
identity, even more preferably 85% identity, even more preferably
90% identity, even more preferably 95% identity, and most
preferably 100% identity) with amino acids 1 to 40 of anyone of SEQ
ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID
NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274, preferably
anyone of SEQ ID NO: 3 to SEQ ID NO: 117 or anyone of SEQ ID NO:
226 to SEQ ID NO: 251 or anyone of SEQ ID
[0072] NO: 252 to SEQ ID NO: 274.
[0073] The term "anyone of SEQ ID NO: 3 to SEQ ID NO: 117" is
intended to mean SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:
15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ
ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:
33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ
ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ
ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:
51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ
ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:
60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:
69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ
ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:
78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ
ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO:
87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ
ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:
96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100,
SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ
ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:
113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO:
117.
[0074] The term "anyone of SEQ ID NO: 118 to SEQ ID NO: 225" is
intended to mean anyone of SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID
NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:
124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:
128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:
132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:
136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:
140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:
144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO:
148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:
152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO:
156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO:
160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO:
164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:
168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO:
172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO:
176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO:
180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:
184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO:
188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:
192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:
196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO:
200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:
204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO:
208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:
212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO:
216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO:
220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO:
224, or SEQ ID NO: 225.
[0075] The term "anyone of SEQ ID NO: 226 to SEQ ID NO: 251" is
intended to mean anyone of SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID
NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO:
232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO:
236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO:
240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO:
244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO:
248, SEQ ID NO: 249, SEQ ID NO: 250, or SEQ ID NO: 251.
[0076] The term "anyone of SEQ ID NO: 252 to SEQ ID NO: 274" is
intended to mean anyone of SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID
NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO:
258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO:
262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO:
266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO:
270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, or SEQ ID NO:
274.
[0077] The term "anyone of SEQ ID NO: 3 to SEQ ID NO: 225" is
intended to mean anyone of SEQ ID NO: 3 to SEQ ID NO: 117 or anyone
of SEQ ID NO: 118 to SEQ ID NO: 225.
[0078] The term "anyone of SEQ ID NO: 2 to SEQ ID NO: 225" is
intended to mean SEQ ID NO: 2 or anyone of SEQ ID NO: 3 to SEQ ID
NO: 225.
[0079] The amino acids making up the polypeptides of the invention
may independently be selected from D or L forms. Preferably the
polypeptide of the invention is a defensin polypeptide; more
preferably an alpha defensin, a beta defensin, or an insect
(arthropod) defensin.
[0080] The polypeptides of the invention may exhibit higher or at
least equal, preferably higher, antimicrobial activity compared to
the polypeptide of SEQ ID NO: 1, determined as the Minimum
Inhibitory Concentration (MIC), against Staphylococcus carnosus
ATCC51365, Staphylococcus aureus ATCC29213 or Staphylococcus aureus
ATCC25923 according to the NCCLS/CLSI guidelines, Protocol M7-A6,
Vol. 20, No. 2: Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria That Grow Aerobically.
[0081] In an embodiment, the present invention relates to
artificial variants comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of anyone of
SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ
ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274.
Preferably, amino acid changes are of a minor nature, that is
conservative amino acid substitutions or insertions that do not
significantly affect the folding and/or activity of the protein;
small deletions, typically of one to about 10 amino acids; small
amino- or carboxyl-terminal extensions, such as an amino-terminal
methionine residue; a small linker peptide of up to about 20-25
residues; or a small extension that facilitates purification by
changing net charge or another function, such as a poly-histidine
tract, an antigenic epitope or a binding domain.
[0082] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions which
do not generally alter specific activity are known in the art and
are described, for example, by H. Neurath and R. L. Hill, 1979, In,
The Proteins, Academic Press, New York. The most commonly occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
[0083] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be
substituted for amino acid residues of a wild-type polypeptide. A
limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, and unnatural amino acids may
be substituted for amino acid residues. "Unnatural amino acids"
have been modified after protein synthesis, and/or have a chemical
structure in their side chain(s) different from that of the
standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and
include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0084] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0085] Essential amino acids in the parent polypeptide can be
identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter
technique, single alanine mutations are introduced at every residue
in the molecule, and the resultant mutant molecules are tested for
biological activity (i.e., antimicrobial activity) to identify
amino acid residues that are critical to the activity of the
molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271:
4699-4708. The biological interaction can also be determined by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction,
or photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids. See, for example, de Vos et al., 1992,
Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:
899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The
identities of essential amino acids can also be inferred from
analysis of identities with polypeptides which are related to a
polypeptide according to the invention.
[0086] Single or multiple amino acid substitutions can be made and
tested using known methods of mutagenesis, recombination, and/or
shuffling, followed by a relevant screening procedure, such as
those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:
53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:
2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be
used include error-prone PCR, phage display (e.g., Lowman et al.,
1991, Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO
92/06204), and region-directed mutagenesis (Derbyshire et al.,
1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
[0087] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells.
Mutagenized DNA molecules that encode active polypeptides can be
recovered from the host cells and rapidly sequenced using standard
methods in the art. These methods allow the rapid determination of
the importance of individual amino acid residues in a polypeptide
of interest, and can be applied to polypeptides of unknown
structure.
[0088] The total number of amino acid substitutions, deletions
and/or insertions of amino acids 1 to 40 of anyone of SEQ ID NO: 2
to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or
anyone of SEQ ID NO: 252 to SEQ ID NO: 274 is 10, preferably 9,
more preferably 8, more preferably 7, more preferably at most 6,
more preferably at most 5, more preferably 4, even more preferably
3, most preferably 2, and even most preferably 1.
[0089] In another aspect, the present invention provides a variant
of a parent antimicrobial polypeptide, which parent antimicrobial
polypeptide has at least 60% identity with the amino acid sequence
of SEQ ID NO: 1, wherein the variant has antimicrobial activity and
comprises a substitution in one or more positions, and wherein the
substitutions are selected from:
F2L, F2W or F2I;
N5R, N5Q, N5V, N5G, N5S, N5A, N5K, N5L, N5M, N5D, N5H or N5Y;
G6R, G6A or G6K;
P7A, P7L, P7V, P7K or P7R;
W8R;
D9A, D9G, D9K, D9L, D9T, D9N, D9F, D9H, D9M, D9P, D9Q, D9S, D9C,
D91, D9R, D9V or D9Y;
E10G, E10A, E10L, E10C, E10Q or E10S;
D11F, D11G, D11N, D11V, D11Y, D11H, D11K, D11L, D11P, D115, D11T,
D11W, D11I, D11M, D11A, D11C or D11R;
D12P;
M13R, M13S, M13V, M13A, M13F, M13G, M13L, M13T, M13Y, M13W, M13E or
M13K;
Q14R, Q14L, Q14F, Q14G, Q14H, Q14S, Q14A, Q14C, Q14I, Q14K, Q14M,
Q14P, Q14T, Q14V, Q14W or Q14Y;
N17R, N17I, N17Y, N17V, N17K, N17T, N17S, N17Q, N17F, N17A, N17W,
N17E or N17H;
H18A, H18F, H18Q, H18T, H18V or H18L;
K20Q or K20R;
S21A, S21V, S21N or S21F;
I22L, I22M, I22T, I22W or I22V;
K23R or K23T;
G24H, G24K, G24A, G24P, G24F, G241, G24Q, G24R, G24S, G24T, G24Y or
G24N;
Y25H, Y25K, Y25L, Y25M, Y25N, Y25Q, Y25S, Y25V or Y25R;
K26F, K26H, K26T, K26C or K26R;
Y29F, Y29R, Y29A, Y29H, Y29L, Y29M, Y29S or Y29W;
A31K, A31N, A31Q, A31T, A31E, A31H, A31I, A31R, A31S, A31V, A31G or
A31Y;
K32R or K32T;
G33K, G33Q, G33E, G33N, G33S, G33T, G33A or G33R;
G34K, G34H, G34W or G34R;
F35A, F35H, F35I, F35M, F35V, F35W, F35R or F35L;
V36L, V36M, V361, V36K, V36Q, V36R or V36T;
K38H, K38N or K38R; and
Y40I, Y40YRCG or Y40YR.
[0090] Preferably the parent antimicrobial polypeptide has at least
70% identity, more preferably at least 80% identity, even more
preferably at least 90% identity, and most preferably 95% identity
with the amino acid sequence of SEQ ID NO: 1. In particular the
parent antimicrobial polypeptide may be identical to the amino acid
sequence of SEQ ID NO: 1.
[0091] In an embodiment the parent antimicrobial polypeptide is a
defensin polypeptide; more preferably an alpha defensin, a beta
defensin, or an insect (arthropod) defensin. Preferably the parent
amino acid sequence exhibit antimicrobial activity.
[0092] In another embodiment, the parent antimicrobial polypeptide
has 1, 2, 3, 4, 5, 6, 7 or 8 amino acid differences compared to the
amino acid sequence of SEQ ID NO: 1. Preferably 1, 2, 3, 4, 5 or 6;
more preferably 1, 2, 3, 4 or 5; even more preferably 1, 2, 3 or 4;
even more preferably 1, 2 or 3; and most preferably 1 or 2 amino
acids are different compared to the amino acid sequence of SEQ ID
NO: 1.
N-Terminal Extension
[0093] An N-terminal extension of the polypeptides of the invention
may suitably consist of from 1 to 50 amino acids, preferably 2-20
amino acids, especially 3-15 amino acids. In one embodiment
N-terminal peptide extension does not contain an Arg (R). In
another embodiment the N-terminal extension comprises a kex2 or
kex2-like cleavage site as will be defined further below. In a
preferred embodiment the N-terminal extension is a peptide,
comprising at least two Glu (E) and/or Asp (D) amino acid residues,
such as an N-terminal extension comprising one of the following
sequences: EAE, EE, DE and DD.
Kex2 Sites
[0094] Kex2 sites (see, e.g., Methods in Enzymology Vol. 185, ed.
D. Goeddel, Academic Press Inc. (1990), San Diego, Calif., "Gene
Expression Technology") and kex2-like sites are di-basic
recognition sites (i.e., cleavage sites) found between the
pro-peptide encoding region and the mature region of some
proteins.
[0095] Insertion of a kex2 site or a kex2-like site have in certain
cases been shown to improve correct endopeptidase processing at the
pro-peptide cleavage site resulting in increased protein secretion
levels.
[0096] In the context of the invention insertion of a kex2 or
kex2-like site result in the possibility to obtain cleavage at a
certain position in the N-terminal extension resulting in an
antimicrobial polypeptide being extended in comparison to amino
acids 1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone
of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to
SEQ ID NO: 274.
Fused Polypeptides
[0097] The polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide of the invention or a fragment thereof. A fused
polypeptide is produced by fusing a nucleotide sequence (or a
portion thereof) encoding another polypeptide to a nucleotide
sequence (or a portion thereof) of the present invention.
Techniques for producing fusion polypeptides are known in the art,
and include ligating the coding sequences encoding the polypeptides
so that they are in frame and that expression of the fused
polypeptide is under control of the same promoter(s) and
terminator.
Sources of Polypeptides Having Antimicrobial Activity
[0098] A polypeptide of the present invention may be obtained from
microorganisms of any genus. For purposes of the present invention,
the term "obtained from" as used herein in connection with a given
source shall mean that the polypeptide encoded by a nucleotide
sequence is produced by the source or by a strain in which the
nucleotide sequence from the source has been inserted. In a
preferred aspect, the polypeptide obtained from a given source is
secreted extracellularly.
[0099] A polypeptide of the present invention may be a bacterial
polypeptide. For example, the polypeptide may be a gram positive
bacterial polypeptide such as a Bacillus polypeptide, e.g., a
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,
Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus
lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus
stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis
polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces
lividans or Streptomyces murinus polypeptide; or a gram negative
bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.
polypeptide.
[0100] A polypeptide of the present invention may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide; or more preferably a filamentous fungal
polypeptide such as an Acremonium, Aspergillus, Aureobasidium,
Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus,
Thielavia, Tolypocladium, or Trichoderma polypeptide.
[0101] In a preferred aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having antimicrobial activity.
[0102] In another preferred aspect, the polypeptide is an
Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,
Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Fusarium bacteridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,
Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,
Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,
Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma
reesei, or Trichoderma viride polypeptide.
[0103] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0104] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0105] Polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding
another polypeptide to a nucleotide sequence (or a portion thereof)
of the present invention. Techniques for producing fusion
polypeptides are known in the art, and include ligating the coding
sequences encoding the polypeptides so that they are in frame and
that expression of the fused polypeptide is under control of the
same promoter(s) and terminator.
Polynucleotides
[0106] The present invention also relates to polynucleotides having
a nucleotide sequence which encodes for a polypeptide of the
invention. In particular, the present invention relates to
polynucleotides consisting of a nucleotide sequence which encodes
for a polypeptide of the invention. Due to the degeneracy of the
genetic code, the skilled person will easily recognize that several
nucleotide sequences encoding each of the polypeptides of the
invention may be prepared. It is well known in the art which
nucleotides make up codons encoding the amino acids of the
polypeptides of the invention.
[0107] The present invention also relates to polynucleotides which
encode fragments of the amino acid sequence shown as anyone of SEQ
ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID
NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274 that have
antimicrobial activity. A subsequence of the polynucleotides is a
nucleotide sequence wherein one or more nucleotides from the 5'
and/or 3' end have been deleted.
[0108] The nucleotide sequence may be obtained by standard cloning
procedures used in genetic engineering to relocate the nucleotide
sequence from one location to a different site where it will be
reproduced. The cloning procedures may involve excision and
isolation of a desired fragment comprising the nucleotide sequence
encoding the polypeptide, insertion of the fragment into a vector
molecule, and incorporation of the recombinant vector into a host
cell where multiple copies or clones of the nucleotide sequence
will be replicated. The nucleotide sequence may be of genomic,
cDNA, RNA, semisynthetic, synthetic origin, or any combinations
thereof.
[0109] Modification of a nucleotide sequence encoding a polypeptide
of the present invention may be necessary for the synthesis of a
polypeptide, which comprises an amino acid sequence that has at
least one substitution, deletion and/or insertion as compared to
amino acids 1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or
anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO:
252 to SEQ ID NO: 274. These artificial variants may differ in some
engineered way from the polypeptide isolated from its native
source, e.g., variants that differ in specific activity,
thermostability, pH optimum, or the like.
[0110] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by an isolated polynucleotide of the invention, and
therefore preferably not subject to substitution, may be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells, 1989, Science 244: 1081-1085). In the latter technique,
mutations are introduced at every positively charged residue in the
molecule, and the resultant mutant molecules are tested for
antimicrobial activity to identify amino acid residues that are
critical to the activity of the molecule. Sites of interaction can
also be determined by analysis of the three-dimensional structure
as determined by such techniques as nuclear magnetic resonance
analysis, crystallography or photoaffinity labelling (see, e.g., de
Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal
of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS
Letters 309: 59-64).
[0111] Moreover, a nucleotide sequence encoding a polypeptide of
the present invention may be modified by introduction of nucleotide
substitutions which do not give rise to another amino acid sequence
of the polypeptide encoded by the nucleotide sequence, but which
correspond to the codon usage of the host organism intended for
production of the antimicrobial polypeptide.
Nucleic Acid Constructs
[0112] The present invention also relates to nucleic acid
constructs comprising an isolated polynucleotide of the present
invention operably linked to one or more control sequences which
direct the expression of the coding sequence in a suitable host
cell under conditions compatible with the control sequences.
[0113] An isolated polynucleotide encoding a polypeptide of the
present invention may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide's sequence prior to its insertion into a vector may
be desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotide sequences utilizing
recombinant DNA methods are well known in the art.
[0114] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence which is recognized by a host cell
for expression of a polynucleotide encoding a polypeptide of the
present invention. The promoter sequence contains transcriptional
control sequences which mediate the expression of the polypeptide.
The promoter may be any nucleotide sequence which shows
transcriptional activity in the host cell of choice including
mutant, truncated, and hybrid promoters, and may be obtained from
genes encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host cell.
[0115] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the E. coli lac operon, Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xylA and xylB genes,
and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731),
as well as the tac promoter (DeBoer et al., 1983, Proceedings of
the National Academy of Sciences USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0116] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei endoglucanase I,
Trichoderma reesei endoglucanase II, Trichoderma reesei
endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma
reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma
reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as
the NA2-tpi promoter (a hybrid of the promoters from the genes for
Aspergillus niger neutral alpha-amylase and Aspergillus oryzae
triose phosphate isomerase); and mutant, truncated, and hybrid
promoters thereof.
[0117] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate
isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1),
and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other
useful promoters for yeast host cells are described by Romanos et
al., 1992, Yeast 8: 423-488.
[0118] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0119] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0120] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0121] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleotide sequence encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used in the present invention.
[0122] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0123] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0124] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleotide
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0125] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0126] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0127] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a host cell of
choice may be used in the present invention. Effective signal
peptide coding regions for bacterial host cells are the signal
peptide coding regions obtained from the genes for Bacillus NCIB
11837 maltogenic amylase, Bacillus stearothermophilus
alpha-amylase, Bacillus licheniformis subtilisin, Bacillus
licheniformis beta-lactamase, Bacillus stearothermophilus neutral
proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further
signal peptides are described by Simonen and Palva, 1993,
Microbiological Reviews 57: 109-137.
[0128] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and
Humicola lanuginosa lipase.
[0129] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
[0130] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0131] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0132] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used as regulatory sequences. Other examples of
regulatory sequences are those which allow for gene amplification.
In eukaryotic systems, these include the dihydrofolate reductase
gene which is amplified in the presence of methotrexate, and the
metallothionein genes which are amplified with heavy metals. In
these cases, the nucleotide sequence encoding the polypeptide would
be operably linked with the regulatory sequence.
Expression Vectors
[0133] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleic acids and control sequences described above may be
joined together to produce a recombinant expression vector which
may include one or more convenient restriction sites to allow for
insertion or substitution of the nucleotide sequence encoding the
polypeptide at such sites. Alternatively, a nucleotide sequence of
the present invention may be expressed by inserting the nucleotide
sequence or a nucleic acid construct comprising the sequence into
an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
[0134] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about expression of the
nucleotide sequence. The choice of the vector will typically depend
on the compatibility of the vector with the host cell into which
the vector is to be introduced. The vectors may be linear or closed
circular plasmids.
[0135] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0136] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like.
[0137] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers which
confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol, or tetracycline resistance. Suitable markers for
yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS and pyrG genes of Aspergillus nidulans or
Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
[0138] The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host
cell's genome or autonomous replication of the vector in the cell
independent of the genome.
[0139] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or nonhomologous recombination. Alternatively, the
vector may contain additional nucleotide sequences for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should preferably contain a sufficient number of nucleic
acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000
base pairs, and most preferably 800 to 10,000 base pairs, which
have a high degree of identity with the corresponding target
sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous
with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
encoding nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0140] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication which functions in a cell. The term "origin of
replication" or "plasmid replicator" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0141] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM31 permitting replication in Bacillus.
[0142] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0143] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67;
Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0144] More than one copy of a polynucleotide of the present
invention may be inserted into the host cell to increase production
of the gene product. An increase in the copy number of the
polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0145] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0146] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising a polynucleotide of the present
invention is introduced into a host cell so that the vector is
maintained as a chromosomal integrant or as a self-replicating
extra-chromosomal vector as described earlier. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication. The
choice of a host cell will to a large extent depend upon the gene
encoding the polypeptide and its source.
[0147] The host cell may be a unicellular microorganism, e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a
eukaryote.
[0148] Useful unicellular microorganisms are bacterial cells such
as gram positive bacteria including, but not limited to, a Bacillus
cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus
coagulans, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces
cell, e.g., Streptomyces lividans and Streptomyces murinus, or gram
negative bacteria such as E. coli and Pseudomonas sp. In a
preferred aspect, the bacterial host cell is a Bacillus lentus,
Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus
subtilis cell. In another preferred aspect, the Bacillus cell is an
alkalophilic Bacillus.
[0149] The introduction of a vector into a bacterial host cell may,
for instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115),
using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0150] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0151] In a preferred aspect, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0152] In a more preferred aspect, the fungal host cell is a yeast
cell. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, Passmore, and Davenport, eds, Soc. App. Bacteriol.
Symposium Series No. 9, 1980).
[0153] In an even more preferred aspect, the yeast host cell is a
Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0154] In a most preferred aspect, the yeast host cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
In another most preferred aspect, the yeast host cell is a
Kluyveromyces lactis cell. In another most preferred aspect, the
yeast host cell is a Yarrowia lipolytica cell.
[0155] In another more preferred aspect, the fungal host cell is a
filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0156] In an even more preferred aspect, the filamentous fungal
host cell is an Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus,
Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, or Trichoderma cell.
[0157] In a most preferred aspect, the filamentous fungal host cell
is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger or Aspergillus oryzae cell. In another most preferred aspect,
the filamentous fungal host cell is a Fusarium bacteridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,
Fusarium trichothecioides, or Fusarium venenatum cell. In another
most preferred aspect, the filamentous fungal host cell is a
Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or
Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,
Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride strain cell.
[0158] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238 023 and Yelton et al., 1984,
Proceedings of the National Academy of Sciences USA 81: 1470-1474.
Suitable methods for transforming Fusarium species are described by
Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast
may be transformed using the procedures described by Becker and
Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to
Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume
194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
[0159] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a cell, which in its wild-type form is capable of producing the
polypeptide, under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0160] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a host cell under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0161] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods well known in the art. For
example, the cell may be cultivated by shake flask cultivation, and
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
[0162] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies. For example, an
antimicrobial activity assay may be used to determine the activity
of the polypeptide as described herein.
[0163] The resulting polypeptide may be recovered using methods
known in the art. For example, the polypeptide may be recovered
from the nutrient medium by conventional procedures including, but
not limited to, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation.
[0164] The polypeptides of the present invention may be purified by
a variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989).
Plants
[0165] The present invention also relates to a transgenic plant,
plant part, or plant cell which has been transformed with a
nucleotide sequence encoding a polypeptide having antimicrobial
activity of the present invention so as to express and produce the
polypeptide in recoverable quantities. The polypeptide may be
recovered from the plant or plant part. Alternatively, the plant or
plant part containing the recombinant polypeptide may be used as
such for improving the quality of a food or feed, e.g., improving
nutritional value, palatability, and rheological properties, or to
destroy an antinutritive factor.
[0166] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0167] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0168] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilisation of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seeds coats.
[0169] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0170] The transgenic plant or plant cell expressing a polypeptide
of the present invention may be constructed in accordance with
methods known in the art. In short, the plant or plant cell is
constructed by incorporating one or more expression constructs
encoding a polypeptide of the present invention into the plant host
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0171] The expression construct is conveniently a nucleic acid
construct which comprises a polynucleotide encoding a polypeptide
of the present invention operably linked with appropriate
regulatory sequences required for expression of the nucleotide
sequence in the plant or plant part of choice. Furthermore, the
expression construct may comprise a selectable marker useful for
identifying host cells into which the expression construct has been
integrated and DNA sequences necessary for introduction of the
construct into the plant in question (the latter depends on the DNA
introduction method to be used).
[0172] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention may be constitutive or inducible, or may be
developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or
leaves. Regulatory sequences are, for example, described by Tague
et al., 1988, Plant Physiology 86: 506.
[0173] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, and the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294, Christensen et al., 1992, Plant Mo.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or
from metabolic sink tissues such as meristems (Ito et al., 1994,
Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba
promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152:
708-711), a promoter from a seed oil body protein (Chen et al.,
1998, Plant and Cell Physiology 39: 935-941), the storage protein
napA promoter from Brassica napus, or any other seed specific
promoter known in the art, e.g., as described in WO 91/14772.
Furthermore, the promoter may be a leaf specific promoter such as
the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant
Physiology 102: 991-1000, the chlorella virus adenine
methyltransferase gene promoter (Mitra and Higgins, 1994, Plant
Molecular Biology 26: 85-93), or the aldP gene promoter from rice
(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674),
or a wound inducible promoter such as the potato pin2 promoter (Xu
et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the
promoter may inducible by abiotic treatments such as temperature,
drought, or alterations in salinity or induced by exogenously
applied substances that activate the promoter, e.g., ethanol,
oestrogens, plant hormones such as ethylene, abscisic acid, and
gibberellic acid, and heavy metals.
[0174] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide of the present invention in the
plant. For instance, the promoter enhancer element may be an intron
which is placed between the promoter and the nucleotide sequence
encoding a polypeptide of the present invention. For instance, Xu
et al., 1993, supra, disclose the use of the first intron of the
rice actin 1 gene to enhance expression.
[0175] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0176] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0177] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 15-38) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994,
Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Molecular Biology 21:
415-428.
[0178] Following transformation, the transformants having
incorporated the expression construct are selected and regenerated
into whole plants according to methods well-known in the art. Often
the transformation procedure is designed for the selective
elimination of selection genes either during regeneration or in the
following generations by using, for example, co-transformation with
two separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0179] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding a polypeptide having antimicrobial activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Compositions
[0180] The present invention also relates to compositions, such as
pharmaceutical compositions, comprising a polypeptide of the
present invention. Preferably, the compositions are enriched in
such a polypeptide. The term "enriched" indicates that the
antimicrobial activity of the composition has been increased, e.g.,
with an enrichment factor of 1.1.
[0181] The compositions may further comprise another
pharmaceutically active agent, such as an additional biocidal or
biostatic agent, such as another antimicrobial polypeptide
exhibiting antimicrobial activity as defined above. The biocidal
agent may be an antibiotic, as known in the art. Classes of
antibiotics include penicillins, e.g., penicillin G, penicillin V,
methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.;
penicillins in combination with beta-lactamase inhibitors,
cephalosporins, e.g., cefaclor, cefazolin, cefuroxime, moxalactam,
etc.; carbapenems; monobactams; aminoglycosides; tetracyclines;
macrolides; lincomycins; polymyxins; sulfonamides; quinolones;
cloramphenical; metronidazole; spectinomycin; trimethoprim;
vancomycin; etc. The biocidal agent may also be an anti-mycotic
agent, including polyenes, e.g., amphotericin B, nystatin;
5-flucosyn; and azoles, e.g., miconazol, ketoconazol, itraconazol
and fluconazol.
[0182] In an embodiment the biocidal agent is a non-enzymatic
chemical agent. In another embodiment the biocidal agent is a
non-polypeptide chemical agent.
[0183] The compositions may comprise a suitable carrier material.
The compositions may also comprise a suitable delivery vehicle
capable of delivering the antimicrobial polypeptides of the
invention to the desired locus when the compositions are used as a
medicament.
[0184] The polypeptide compositions may be prepared in accordance
with methods known in the art and may be in the form of a liquid or
a dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art. Examples are given below of preferred
uses of the polypeptide compositions of the invention. The dosage
of the polypeptide composition of the invention and other
conditions under which the composition is used may be determined on
the basis of methods known in the art.
Methods and Uses
[0185] The present invention is also directed to methods for using
the polypeptides having antimicrobial activity. The antimicrobial
polypeptides are typically useful at any locus subject to
contamination by bacteria, fungi, yeast or algae. Typically, loci
are in aqueous systems such as cooling water systems, laundry rinse
water, oil systems such as cutting oils, lubricants, oil fields and
the like, where microorganisms need to be killed or where their
growth needs to be controlled. However, the present invention may
also be used in all applications for which known antimicrobial
compositions are useful, such as protection of wood, latex,
adhesive, glue, paper, cardboard, textile, leather, plastics,
caulking, and feed.
[0186] Other uses include preservation of foods, beverages,
cosmetics such as lotions, creams, gels, ointments, soaps,
shampoos, conditioners, antiperspirants, deodorants, mouth wash,
contact lens products, enzyme formulations, or food
ingredients.
[0187] Thus, the antimicrobial polypeptides of the invention may by
useful as a disinfectant, e.g., in the treatment of infections in
the eye or the mouth, skin infections; in antiperspirants or
deodorants; for cleaning and disinfection of contact lenses and
teeth (oral care).
[0188] In general it is contemplated that the antimicrobial
polypeptides of the present invention are useful for cleaning,
disinfecting or inhibiting microbial growth on any surface.
Examples of surfaces, which may advantageously be contacted with
the antimicrobial polypeptides of the invention are surfaces of
process equipment used, e.g., dairies, chemical or pharmaceutical
process plants, water sanitation systems, oil processing plants,
paper pulp processing plants, water treatment plants, and cooling
towers. The antimicrobial polypeptides of the invention should be
used in an amount, which is effective for cleaning, disinfecting or
inhibiting microbial growth on the surface in question.
[0189] The antimicrobial polypeptides of the invention may
additionally be used for cleaning surfaces and cooking utensils in
food processing plants and in any area in which food is prepared or
served such as hospitals, nursing homes and restaurants.
[0190] It may also be used as a preservation agent or a
disinfection agent in water based paints.
[0191] The invention also relates to the use of an antimicrobial
polypeptide or composition of the invention as a medicament.
Further, an antimicrobial polypeptide or composition of the
invention may also be used for the manufacture of a medicament for
controlling or combating microorganisms, such as fungal organisms
or bacteria, preferably gram positive bacteria.
[0192] The composition and antimicrobial polypeptide of the
invention may be used as an antimicrobial veterinarian or human
therapeutic or prophylactic agent. Thus, the composition and
antimicrobial polypeptide of the invention may be used in the
preparation of veterinarian or human therapeutic agents or
prophylactic agents for the treatment of microbial infections, such
as bacterial or fungal infections, preferably gram positive
bacterial infections. In particular the microbial infections may be
associated with lung diseases including, but not limited to,
tuberculosis, pneumonia and cystic fibrosis; and sexual transmitted
diseases including, but not limited to, gonorrhea and
chlamydia.
[0193] The composition of the invention comprises an effective
amount of the antimicrobial polypeptide of the invention.
[0194] The term "effective amount" when used herein is intended to
mean an amount of the antimicrobial polypeptides of the invention,
which is sufficient to inhibit growth of the microorganisms in
question.
[0195] The invention also relates to wound healing compositions or
products such as bandages, medical devices such as, e.g., catheters
and further to anti-dandruff hair products, such as shampoos.
[0196] Formulations of the antimicrobial polypeptides of the
invention are administered to a host suffering from or predisposed
to a microbial infection. Administration may be topical, localized
or systemic, depending on the specific microorganism, preferably it
will be localized. Generally the dose of the antimicrobial
polypeptides of the invention will be sufficient to decrease the
microbial population by at least about 50%, usually by at least 1
log, and may be by 2 or more logs of killing. The compounds of the
present invention are administered at a dosage that reduces the
microbial population while minimizing any side-effects. It is
contemplated that the composition will be obtained and used under
the guidance of a physician for in vivo use. The antimicrobial
polypeptides of the invention are particularly useful for killing
gram negative bacteria, including Pseudomonas aeruginosa, and
Chlamydia trachomatis; and gram-positive bacteria, including
streptococci such as Streptococcus pneumonia, S. uberis, S.
hyointestinalis, S. pyogenes and S. agalactiae; and staphylococci
such as Staphylococcus aureus, S. epidermidis, S. simulans, S.
xylosus and S. carnosus.
[0197] Formulations of the antimicrobial polypeptides of the
invention may be administered to a host suffering from or
predisposed to a microbial lung infection, such as pneumonia; or to
a microbial wound infection, such as a bacterial wound
infection.
[0198] Formulations of the antimicrobial polypeptides of the
invention may also be administered to a host suffering from or
predisposed to a skin infection, such as acne, atopic dermatitis or
seborrheic dermatitis; preferably the skin infection is a bacterial
skin infection, e.g., caused by Staphylococcus epidermidis,
Staphylococcus aureus, Propionibacterium acnes, Pityrosporum ovale
or Malassezia furfur.
[0199] The antimicrobial polypeptides of the invention are also
useful for in vitro formulations to kill microbes, particularly
where one does not wish to introduce quantities of conventional
antibiotics. For example, the antimicrobial polypeptides of the
invention may be added to animal and/or human food preparations; or
they may be included as an additive for in vitro cultures of cells,
to prevent the overgrowth of microbes in tissue culture.
[0200] The susceptibility of a particular microbe to killing with
the antimicrobial polypeptides of the invention may be determined
by in vitro testing, as detailed in the experimental section.
Typically a culture of the microbe is combined with the
antimicrobial polypeptide at varying concentrations for a period of
time sufficient to allow the protein to act, usually between about
one hour and one day. The viable microbes are then counted, and the
level of killing determined.
[0201] Microbes of interest include, but are not limited to,
Gram-negative bacteria, for example: Citrobacter sp.; Enterobacter
sp.; Escherichia sp., e.g., E. coli; Klebsiella sp.; Morganella
sp.; Proteus sp.; Providencia sp.; Salmonella sp., e.g., S. typhi,
S. typhimurium; Serratia sp.; Shigella sp.; Pseudomonas sp., e.g.,
P. aeruginosa; Yersinia sp., e.g., Y. pestis, Y.
pseudotuberculosis, Y. enterocolitica; Franciscella sp.; Pasturella
sp.; Vibrio sp., e.g., V. cholerae, V. parahemolyticus;
Campylobacter sp., e.g., C. jejuni; Haemophilus sp., e.g., H.
influenzae, H. ducreyi; Bordetella sp., e.g., B. pertussis, B.
bronchiseptica, B. parapertussis; Brucella sp., Neisseria sp.,
e.g., N. gonorrhoeae, N. meningitidis, etc. Other bacteria of
interest include Legionella sp., e.g., L. pneumophila; Listeria
sp., e.g., L. monocytogenes; Mycoplasma sp., e.g., M. hominis, M.
pneumoniae; Mycobacterium sp., e.g., M. tuberculosis, M. leprae;
Treponema sp., e.g., T. pallidum; Borrelia sp., e.g., B.
burgdorferi; Leptospirae sp.; Rickettsia sp., e.g., R. rickettsii,
R. typhi; Chlamydia sp., e.g., C. trachomatis, C. pneumoniae, C.
psittaci; Helicobacter sp., e.g., H. pylori, etc.
[0202] Non-bacterial pathogens of interest include fungal and
protozoan pathogens, e.g., Plasmodia sp., e.g., P. falciparum,
Trypanosoma sp., e.g., T. brucei; shistosomes; Entaemoeba sp.,
Cryptococcus sp., Candida sp., e.g., C. albicans; etc.
[0203] Various methods for administration may be employed. The
polypeptide formulation may be given orally, or may be injected
intravascularly, subcutaneously, peritoneally, by aerosol,
opthalmically, intra-bladder, topically, etc. For example, methods
of administration by inhalation are well-known in the art. The
dosage of the therapeutic formulation will vary widely, depending
on the specific antimicrobial polypeptide to be administered, the
nature of the disease, the frequency of administration, the manner
of administration, the clearance of the agent from the host, and
the like. The initial dose may be larger, followed by smaller
maintenance doses. The dose may be administered as infrequently as
weekly or biweekly, or fractionated into smaller doses and
administered once or several times daily, semi-weekly, etc. to
maintain an effective dosage level. In many cases, oral
administration will require a higher dose than if administered
intravenously. The amide bonds, as well as the amino and carboxy
termini, may be modified for greater stability on oral
administration. For example, the carboxy terminus may be
amidated.
Formulations
[0204] The compounds of this invention can be incorporated into a
variety of formulations for therapeutic administration. More
particularly, the compounds of the present invention can be
formulated into pharmaceutical compositions by combination with
appropriate, pharmaceutically acceptable carriers or diluents, and
may be formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, creams, foams, solutions, suppositories, injections,
inhalants, gels, microspheres, lotions, and aerosols. As such,
administration of the compounds can be achieved in various ways,
including oral, buccal, rectal, parenteral, intraperitoneal,
intradermal, transdermal, intratracheal, etc., administration. The
antimicrobial polypeptides of the invention may be systemic after
administration or may be localized by the use of an implant or
other formulation that acts to retain the active dose at the site
of implantation.
[0205] In one embodiment, a formulation for topical use comprises a
chelating agent that decreases the effective concentration of
divalent cations, particularly calcium and magnesium. For example,
agents such as citrate, EGTA or EDTA may be included, where citrate
is preferred. The concentration of citrate will usually be from
about 1 to 10 mM.
[0206] The compounds of the present invention can be administered
alone, in combination with each other, or they can be used in
combination with other known compounds (e.g., perforin,
anti-inflammatory agents, antibiotics, etc.). In pharmaceutical
dosage forms, the compounds may be administered in the form of
their pharmaceutically acceptable salts. The following methods and
excipients are merely exemplary and are in no way limiting.
[0207] For oral preparations, the compounds can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0208] The compounds can be formulated into preparations for
injections by dissolving, suspending or emulsifying them in an
aqueous or nonaqueous solvent, such as vegetable or other similar
oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0209] The compounds can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0210] The compounds can be used as lotions, for example to prevent
infection of burns, by formulation with conventional additives such
as solubilizers, isotonic agents, suspending agents, emulsifying
agents, stabilizers and preservatives.
[0211] Furthermore, the compounds can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0212] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more compounds of the present invention.
Similarly, unit dosage forms for injection or intravenous
administration may comprise the compound of the present invention
in a composition as a solution in sterile water, normal saline or
another pharmaceutically acceptable carrier.
[0213] Implants for sustained release formulations are well-known
in the art. Implants are formulated as microspheres, slabs, etc.
with biodegradable or non-biodegradable polymers. For example,
polymers of lactic acid and/or glycolic acid form an erodible
polymer that is well-tolerated by the host. The implant containing
the antimicrobial polypeptides of the invention is placed in
proximity to the site of infection, so that the local concentration
of active agent is increased relative to the rest of the body.
[0214] The term "unit dosage form", as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the unit dosage forms of the present invention
depend on the particular compound employed and the effect to be
achieved, and the pharmacodynamics associated with the compound in
the host.
[0215] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0216] Typical dosages for systemic administration range from 0.1
pg to 100 milligrams per kg weight of subject per administration. A
typical dosage may be one tablet taken from two to six times daily,
or one time-release capsule or tablet taken once a day and
containing a proportionally higher content of active ingredient.
The time-release effect may be obtained by capsule materials that
dissolve at different pH values, by capsules that release slowly by
osmotic pressure, or by any other known means of controlled
release.
[0217] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Some of the specific compounds are more potent than others.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means. A preferred means
is to measure the physiological potency of a given compound.
[0218] The use of liposomes as a delivery vehicle is one method of
interest. The liposomes fuse with the cells of the target site and
deliver the contents of the lumen intracellularly. The liposomes
are maintained in contact with the cells for sufficient time for
fusion, using various means to maintain contact, such as isolation,
binding agents, and the like. In one aspect of the invention,
liposomes are designed to be aerosolized for pulmonary
administration. Liposomes may be prepared with purified proteins or
peptides that mediate fusion of membranes, such as Sendai virus or
influenza virus, etc. The lipids may be any useful combination of
known liposome forming lipids, including cationic or zwitterionic
lipids, such as phosphatidylcholine. The remaining lipid will be
normally neutral or acidic lipids, such as cholesterol,
phosphatidyl serine, phosphatidyl glycerol, and the like.
[0219] For preparing the liposomes, the procedure described by Kato
et al., 1991, J. Biol. Chem. 266: 3361 may be used. Briefly, the
lipids and lumen composition containing peptides are combined in an
appropriate aqueous medium, conveniently a saline medium where the
total solids will be in the range of about 1-10 weight percent.
After intense agitation for short periods of time, from about 5-60
sec., the tube is placed in a warm water bath, from about
25-40.degree. C. and this cycle repeated from about 5-10 times. The
composition is then sonicated for a convenient period of time,
generally from about 1-10 sec. and may be further agitated by
vortexing. The volume is then expanded by adding aqueous medium,
generally increasing the volume by about from 1-2 fold, followed by
shaking and cooling. This method allows for the incorporation into
the lumen of high molecular weight molecules.
Formulations with Other Active Agents
[0220] For use in the subject methods, the antimicrobial
polypeptides of the invention may be formulated with other
pharmaceutically active agents, particularly other antimicrobial
agents. Other agents of interest include a wide variety of
antibiotics, as known in the art. Classes of antibiotics include
penicillins, e.g., penicillin G, penicillin V, methicillin,
oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins
in combination with beta-lactamase inhibitors, cephalosporins,
e.g., cefaclor, cefazolin, cefuroxime, moxalactam, etc.;
carbapenems; monobactams; aminoglycosides; tetracyclines;
macrolides; lincomycins; polymyxins; sulfonamides; quinolones;
cloramphenical; metronidazole; spectinomycin; trimethoprim;
vancomycin; etc.
[0221] Anti-mycotic agents are also useful, including polyenes,
e.g., amphotericin B, nystatin; 5-flucosyn; and azoles, e.g.,
miconazol, ketoconazol, itraconazol and fluconazol.
Antituberculotic drugs include isoniazid, ethambutol, streptomycin
and rifampin. Cytokines may also be included in a formulation of
the antimicrobial polypeptides of the invention, e.g., interferon
gamma, tumor necrosis factor alpha, interleukin 12, etc.
In Vitro Synthesis
[0222] The antimicrobial peptides of the invention may be prepared
by in vitro synthesis, using conventional methods as known in the
art. Various commercial synthetic apparatuses are available, for
example automated synthesizers by Applied Biosystems Inc., Beckman,
etc. By using synthesizers, naturally occurring amino acids may be
substituted with unnatural amino acids, particularly D-isomers (or
D-forms), e.g., D-alanine and D-isoleucine, diastereoisomers, side
chains having different lengths or functionalities, and the like.
The particular sequence and the manner of preparation will be
determined by convenience, economics, purity required, and the
like.
[0223] Chemical linking may be provided to various peptides or
proteins comprising convenient functionalities for bonding, such as
amino groups for amide or substituted amine formation, e.g.,
reductive amination, thiol groups for thioether or disulfide
formation, carboxyl groups for amide formation, and the like.
[0224] If desired, various groups may be introduced into the
peptide during synthesis or during expression, which allow for
linking to other molecules or to a surface. Thus cysteines can be
used to make thioethers, histidines for linking to a metal ion
complex, carboxyl groups for forming amides or esters, amino groups
for forming amides, and the like.
[0225] The polypeptides may also be isolated and purified in
accordance with conventional methods of recombinant synthesis. A
lysate may be prepared of the expression host and the lysate
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, or other purification technique. For the
most part, the compositions which are used will comprise at least
20% by weight of the desired product, more usually at least about
75% by weight, preferably at least about 95% by weight, and for
therapeutic purposes, usually at least about 99.5% by weight, in
relation to contaminants related to the method of preparation of
the product and its purification. Usually, the percentages will be
based upon total protein
Animal Feed
[0226] The present invention is also directed to methods for using
the polypeptides having antimicrobial activity in animal feed, as
well as to feed compositions and feed additives comprising the
antimicrobial polypeptides of the invention.
[0227] The term animal includes all animals, including human
beings. Examples of animals are non-ruminants, and ruminants, such
as cows, sheep and horses. In a particular embodiment, the animal
is a non-ruminant animal. Non-ruminant animals include mono-gastric
animals, e.g., pigs or swine (including, but not limited to,
piglets, growing pigs, and sows); poultry such as turkeys and
chicken (including but not limited to broiler chicks, layers);
young calves; and fish (including but not limited to salmon).
[0228] The term feed or feed composition means any compound,
preparation, mixture, or composition suitable for, or intended for
intake by an animal.
[0229] In the use according to the invention the antimicrobial
polypeptide can be fed to the animal before, after, or
simultaneously with the diet. The latter is preferred.
[0230] In a particular embodiment, the antimicrobial polypeptide,
in the form in which it is added to the feed, or when being
included in a feed additive, is well defined. Well-defined means
that the antimicrobial polypeptide preparation is at least 50% pure
as determined by Size-exclusion chromatography (see Example 12 of
WO 01/58275). In other particular embodiments the antimicrobial
polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94,
or at least 95% pure as determined by this method.
[0231] A well-defined antimicrobial polypeptide preparation is
advantageous. For instance, it is much easier to dose correctly to
the feed an antimicrobial polypeptide that is essentially free from
interfering or contaminating other antimicrobial polypeptides. The
term dose correctly refers in particular to the objective of
obtaining consistent and constant results, and the capability of
optimising dosage based upon the desired effect.
[0232] For the use in animal feed, however, the antimicrobial
polypeptide need not be that pure; it may, e.g., include other
enzymes, in which case it could be termed an antimicrobial
polypeptide preparation.
[0233] The antimicrobial polypeptide preparation can be (a) added
directly to the feed (or used directly in a treatment process of
vegetable proteins), or (b) it can be used in the production of one
or more intermediate compositions such as feed additives or
premixes that is subsequently added to the feed (or used in a
treatment process). The degree of purity described above refers to
the purity of the original antimicrobial polypeptide preparation,
whether used according to (a) or (b) above.
[0234] Antimicrobial polypeptide preparations with purities of this
order of magnitude are in particular obtainable using recombinant
methods of production, whereas they are not so easily obtained and
also subject to a much higher batch-to-batch variation when the
antimicrobial polypeptide is produced by traditional fermentation
methods.
[0235] Such antimicrobial polypeptide preparation may of course be
mixed with other enzymes.
[0236] The term vegetable proteins as used herein refers to any
compound, composition, preparation or mixture that includes at
least one protein derived from or originating from a vegetable,
including modified proteins and protein-derivatives. In particular
embodiments, the protein content of the vegetable proteins is at
least 10, 20, 30, 40, 50, or 60% (w/w).
[0237] Vegetable proteins may be derived from vegetable protein
sources, such as legumes and cereals, for example materials from
plants of the families Fabaceae (Leguminosae), Cruciferaceae,
Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal and
rapeseed meal.
[0238] In a particular embodiment, the vegetable protein source is
material from one or more plants of the family Fabaceae, e.g.,
soybean, lupine, pea, or bean.
[0239] In another particular embodiment, the vegetable protein
source is material from one or more plants of the family
Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa.
[0240] Other examples of vegetable protein sources are rapeseed,
and cabbage.
[0241] Soybean is a preferred vegetable protein source.
[0242] Other examples of vegetable protein sources are cereals such
as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
[0243] The antimicrobial polypeptide can be added to the feed in
any form, be it as a relatively pure antimicrobial polypeptide, or
in admixture with other components intended for addition to animal
feed, i.e., in the form of animal feed additives, such as the
so-called pre-mixes for animal feed.
[0244] In a further aspect the present invention relates to
compositions for use in animal feed, such as animal feed, and
animal feed additives, e.g., premixes.
[0245] Apart from the antimicrobial polypeptide of the invention,
the animal feed additives of the invention contain at least one fat
soluble vitamin, and/or at least one water soluble vitamin, and/or
at least one trace mineral, and/or at least one macro mineral.
[0246] Further, optional, feed-additive ingredients are colouring
agents, aroma compounds, stabilisers, and/or at least one other
enzyme selected from amongst phytases EC 3.1.3.8 or 3.1.3.26;
xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and/or
beta-glucanases EC 3.2.1.4.
[0247] In a particular embodiment these other enzymes are well
defined (as defined above for antimicrobial polypeptide
preparations).
[0248] Examples of other antimicrobial peptides (AMPs) are CAP18,
Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Ovispirin
such as Novispirin (Robert Lehrer, 2000), and variants, or
fragments thereof which retain antimicrobial activity.
[0249] Examples of other antifungal polypeptides (AFPs) are the
Aspergillus giganteus, and Aspergillus niger peptides, as well as
variants and fragments thereof which retain antifungal activity, as
disclosed in WO 94/01459 and WO 02/090384.
[0250] Usually fat and water soluble vitamins, as well as trace
minerals form part of a so-called premix intended for addition to
the feed, whereas macro minerals are usually separately added to
the feed. Either of these composition types, when enriched with an
antimicrobial polypeptide of the invention, is an animal feed
additive of the invention.
[0251] In a particular embodiment, the animal feed additive of the
invention is intended for being included (or prescribed as having
to be included) in animal diets or feed at levels of 0.01 to 10.0%;
more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g
additive per 100 g feed). This is so in particular for
premixes.
[0252] The following are non-exclusive lists of examples of these
components:
[0253] Examples of fat soluble vitamins are vitamin A, vitamin D3,
vitamin E, and vitamin K, e.g., vitamin K3.
[0254] Examples of water soluble vitamins are vitamin B12, biotin
and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid
and panthothenate, e.g., Ca-D-panthothenate.
[0255] Examples of trace minerals are manganese, zinc, iron,
copper, iodine, selenium, and cobalt.
[0256] Examples of macro minerals are calcium, phosphorus and
sodium.
[0257] The nutritional requirements of these components
(exemplified with poultry and piglets/pigs) are listed in Table A
of WO 01/58275. Nutritional requirement means that these components
should be provided in the diet in the concentrations indicated.
[0258] In the alternative, the animal feed additive of the
invention comprises at least one of the individual components
specified in Table A of WO 01/58275. At least one means either of,
one or more of, one, or two, or three, or four and so forth up to
all thirteen, or up to all fifteen individual components. More
specifically, this at least one individual component is included in
the additive of the invention in such an amount as to provide an
in-feed-concentration within the range indicated in column four, or
column five, or column six of Table A.
[0259] The present invention also relates to animal feed
compositions. Animal feed compositions or diets have a relatively
high content of protein. Poultry and pig diets can be characterised
as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can
be characterised as indicated in column 4 of this Table B.
Furthermore such fish diets usually have a crude fat content of
200-310 g/kg.
[0260] An animal feed composition according to the invention has a
crude protein content of 50-800 g/kg, and furthermore comprises at
least one antimicrobial polypeptide as claimed herein.
[0261] Furthermore, or in the alternative (to the crude protein
content indicated above), the animal feed composition of the
invention has a content of metabolizable energy of 10-30 MJ/kg;
and/or a content of calcium of 0.1-200 g/kg; and/or a content of
available phosphorus of 0.1-200 g/kg; and/or a content of
methionine of 0.1-100 g/kg; and/or a content of methionine plus
cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50
g/kg.
[0262] In particular embodiments, the content of metabolisable
energy, crude protein, calcium, phosphorus, methionine, methionine
plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or
5 in Table B of WO 01/58275 (R. 2-5).
[0263] Crude protein is calculated as nitrogen (N) multiplied by a
factor 6.25, i.e., Crude protein (g/kg)=N (g/kg).times.6.25. The
nitrogen content is determined by the Kjeldahl method (A.O.A.C.,
1984, Official Methods of Analysis 14th ed., Association of
Official Analytical Chemists, Washington D.C.).
[0264] Metabolizable energy can be calculated on the basis of the
NRC publication Nutrient requirements in swine, ninth revised
edition 1988, subcommittee on swine nutrition, committee on animal
nutrition, board of agriculture, national research council.
National Academy Press, Washington, D.C., pp. 2-6, and the European
Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre
for poultry research and extension, 7361 DA Beekbergen, The
Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen.
ISBN 90-71463-12-5.
[0265] The dietary content of calcium, available phosphorus and
amino acids in complete animal diets is calculated on the basis of
feed tables such as Veevoedertabel 1997, gegevens over chemische
samenstelling, verteerbaarheid en voederwaarde van voedermiddelen,
Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN
90-72839-13-7.
[0266] In a particular embodiment, the animal feed composition of
the invention contains at least one vegetable protein or protein
source as defined above.
[0267] In still further particular embodiments, the animal feed
composition of the invention contains 0-80% maize; and/or 0-80%
sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30%
oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or
0-20% whey. Animal diets can, e.g., be manufactured as mash feed
(non pelleted) or pelleted feed. Typically, the milled feed-stuffs
are mixed and sufficient amounts of essential vitamins and minerals
are added according to the specifications for the species in
question. Enzymes can be added as solid or liquid enzyme
formulations. For example, a solid enzyme formulation is typically
added before or during the mixing step; and a liquid enzyme
preparation is typically added after the pelleting step. The enzyme
may also be incorporated in a feed additive or premix.
[0268] The final enzyme concentration in the diet is within the
range of 0.01-200 mg enzyme protein per kg diet, for example in the
range of 5-30 mg enzyme protein per kg animal diet.
[0269] The antimicrobial polypeptide may be administered in one or
more of the following amounts (dosage ranges): 0.01-200; or
0.01-100; or 0.05-100; or 0.05-50; or 0.10-10--all these ranges
being in mg antimicrobial polypeptide protein per kg feed
(ppm).
[0270] For determining mg antimicrobial polypeptide protein per kg
feed, the antimicrobial polypeptide is purified from the feed
composition, and the specific activity of the purified
antimicrobial polypeptide is determined using a relevant assay (see
under antimicrobial activity, substrates, and assays). The
antimicrobial activity of the feed composition as such is also
determined using the same assay, and on the basis of these two
determinations, the dosage in mg antimicrobial polypeptide protein
per kg feed is calculated.
[0271] The same principles apply for determining mg antimicrobial
polypeptide protein in feed additives. Of course, if a sample is
available of the antimicrobial polypeptide used for preparing the
feed additive or the feed, the specific activity is determined from
this sample (no need to purify the antimicrobial polypeptide from
the feed composition or the additive).
[0272] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
[0273] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Example 1
Evaluation of Minimal Effective Concentration
[0274] Three antimicrobial polypeptides, which are variants of SEQ
ID NO: 1, were tested for antimicrobial activity. A Minimal
Effective Concentration assay (MEC, expressed as micrograms/mL)
against different microorganisms was performed using the three
plectasin mutants, Y40YR, N17R and Y25R following the protocol
described in the book of Methods in Molecular Biology, Vol. 78,
Antibacterial peptide protocol William M. Shafer, Human Press.
[0275] The plectasin mutant "Y40YR" was constructed by adding an
arginine residue at the end of the plectasin amino acid
sequence.
[0276] The results showed improved activity of all three
antimicrobial peptides compared to wildtype plectasin (SEQ ID NO:
1) against the bacteria Bacillus subtilis, Micrococcus luteus and
Staphylococcus epidermidis.
TABLE-US-00004 TABLE 1 MEC values; all values are micrograms/mL MEC
against MEC against MEC against Mutation SEQ ID NO: B. subtilis M.
luteus S. epidermis wildtype 1 0.09 1.89 0.98 Y40YR 99 0.05 0.18
0.31 N17R 95 0.07 0.32 0.59 Y25R 117 0.04 1.40 ND
Example 2
Evaluation of Antimicrobial Activity
[0277] A range of antimicrobial polypeptides, which are variants of
SEQ ID NO: 1 (Plectasin), were tested for antimicrobial activity by
expressing them in S. cerevisae and screening the supernatant of
the yeast transformants for antimicrobial activity against
Staphylococcus carnosus ATCC51365.
[0278] Growth media and solutions were prepared as described in
Sambrook, Fritsch and Maniatis (1989), Molecular cloning, Cold
Spring Harbour, Laboratory Press, New York.
[0279] Radial Diffusion Assay was carried out as described in
Methods in Molecular Biology, Vol. 78, Antibacterial peptide
protocol, William M. Shafer, Human Press.
[0280] 200-300 yeast transformant colonies were plated on 14 cm
round plates containing 25 mL SC growth medium supplemented with
1.5% galactose, 0.5% glucose and 1.5% agarose. Plates were
incubated for three hours at room temperature, overlaid with 25 mL
of the same growth medium and allowed to grow for three days at
30.degree. C. Then the plates were overlaid with 25 mL of LB growth
medium containing 1.5% agarose and 10.sup.5 cells of the indicator
strain Staphylococcus carnosus and incubated at 30.degree. C.
overnight to allow growth of the bacterial cells. Next day, the
plates were stained with 1.5 mM MTT to facilitate visualization of
the clearing zones.
[0281] Yeast colonies creating clearing zones were transferred to
microtiter plates containing 200 micro-L of SC growth medium
supplemented with 2% glucose and ampicillin (100 mg/L). Such plates
designated "master plates" were incubated for 2 days at 30.degree.
C. with shaking at 450 rpm to allow yeast growth.
[0282] 10 micro-L SC growth medium from each well of the master
plates were transferred to new microtiter plates containing 200
micro-L SC growth medium with 1.5% galactose and 0.5% glucose.
These plates were called daughter plates and were incubated for 3
days at 30.degree. C. under 450 rpm shaking to allow yeast growth
and peptide synthesis.
[0283] Finally a Radial Diffusion Assay (RDA) was performed
following the protocol described in Methods in Molecular Biology to
analyse and quantify the antimicrobial activity of the yeast
supernatants against S. carnosus.
[0284] Briefly, 30 mL of minimal underlay medium containing 1%
agarose and 5.times.10.sup.5 cfu/mL of S. carnosus was poured in an
omnitray plate (Nunc, 242811). A Nunc TSP plate (#445497) was
inserted immediately on the plate to allow a 96 well pattern
formation. Once the media had solidified, the TSP plate was removed
and 10 micro-L of yeast supernatant samples were applied on the
holes. Plates were incubated 3 hours at 37.degree. C. and overlaid
with 15 mL of LB agar growth medium. Finally, plates were incubated
overnight at 37.degree. C. and coloured with 1.5 mM MTT to
visualize the clearing zones.
[0285] Inspection and measurements of the clearing zones was
performed on the plates. The corresponding yeast clones resulting
in clearing zones of similar or increased size than the clones
encoding for wild type plectasin were picked from the master plates
and transferred to agar plates containing SC growth medium with 2%
glucose and ampicillin (100 mg/L). Such plates were incubated for
further 2 days at 30.degree. C. to allow yeast growth.
Subsequently, colony PCR was performed followed by sequence
analysis to identify amino acid changes in the plectasin
sequence.
[0286] The mutations and corresponding antimicrobial activities,
relative to the activity of plectasin, are shown in Table 2. An
activity of 2 corresponds to the activity of Plectasin. An activity
of 3 is better than Plectasin, and 1 is worse than Plectasin.
TABLE-US-00005 TABLE 2 Antimicrobial activity data from the yeast
screening assay SEQ ID Sequence Mutation(s) NO: Activity
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY wildtype 1 2
GFGCRGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5R 3 3
GFGCQGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5Q 4 3
GFGCVGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5V 5 3
GFGCGGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5G 6 3
GFGCSGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5S 7 3
GFGCAGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5A 8 3
GFGCNGKWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY P7K 9 3
GFGCNGRWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY P7R 10 3
GFGCNGPRDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY W8R 11 3
GFGCNGPWAEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9A 12 3
GFGCNGPWGEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9G 13 3
GFGCNGPWKEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9K 14 3
GFGCNGPWLEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9L 15 3
GFGCNGPWTEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9T 16 3
GFGCNGPWYEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9Y 17 3
GFGCNGPWFEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9F 18 3
GFGCNGPWHEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9H 19 3
GFGCNGPWMEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9M 20 3
GFGCNGPWNEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9N 21 3
GFGCNGPWPEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9P 22 3
GFGCNGPWQEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9Q 23 3
GFGCNGPWSEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9S 24 3
GFGCNGPWVEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9V 25 3
GFGCNGPWDGDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY E10G 26 3
GFGCNGPWDSDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY E10S 27 3
GFGCNGPWDEFDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11F 28 3
GFGCNGPWDEGDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11G 29 3
GFGCNGPWDEHDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11H 30 3
GFGCNGPWDEKDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11K 31 3
GFGCNGPWDELDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11L 32 3
GFGCNGPWDEPDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11P 33 3
GFGCNGPWDESDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11S 34 3
GFGCNGPWDETDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11T 35 3
GFGCNGPWDEVDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11V 36 3
GFGCNGPWDEWDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11W 37 3
GFGCNGPWDEIDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11I 38 3
GFGCNGPWDEMDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11M 39 3
GFGCNGPWDENDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11N 40 3
GFGCNGPWDERDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11R 41 3
GFGCNGPWDEYDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11Y 42 3
GFGCNGPWDEDDRQCHNHCKSIKGYKGGYCAKGGFVCKCY M13R 43 3
GFGCNGPWDEDDSQCHNHCKSIKGYKGGYCAKGGFVCKCY M13S 44 3
GFGCNGPWDEDDVQCHNHCKSIKGYKGGYCAKGGFVCKCY M13V 45 3
GFGCNGPWDEDDMFCHNHCKSIKGYKGGYCAKGGFVCKCY Q14F 46 3
GFGCNGPWDEDDMGCHNHCKSIKGYKGGYCAKGGFVCKCY Q14G 47 3
GFGCNGPWDEDDMHCHNHCKSIKGYKGGYCAKGGFVCKCY Q14H 48 3
GFGCNGPWDEDDMSCHNHCKSIKGYKGGYCAKGGFVCKCY Q14S 49 3
GFGCNGPWDEDDMYCHNHCKSIKGYKGGYCAKGGFVCKCY Q14Y 50 3
GFGCNGPWDEDDMQCHNLCKSIKGYKGGYCAKGGFVCKCY H18L 51 3
GFGCNGPWDEDDMQCHNHCKSLKGYKGGYCAKGGFVCKCY I22L 52 3
GFGCNGPWDEDDMQCHNHCKSVKGYKGGYCAKGGFVCKCY I22V 53 3
GFGCNGPWDEDDMQCHNHCKSIKHYKGGYCAKGGFVCKCY G24H 54 3
GFGCNGPWDEDDMQCHNHCKSIKKYKGGYCAKGGFVCKCY G24K 55 3
GFGCNGPWDEDDMQCHNHCKSIKNYKGGYCAKGGFVCKCY G24N 56 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGFCAKGGFVCKCY Y29F 57 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGRCAKGGFVCKCY Y29R 58 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGWCAKGGFVCKCY Y29W 59 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCKKGGFVCKCY A31K 60 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCNKGGFVCKCY A31N 61 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCQKGGFVCKCY A31Q 62 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCTKGGFVCKCY A31T 63 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCYKGGFVCKCY A31Y 64 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKKGFVCKCY G33K 65 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKQGFVCKCY G33Q 66 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKRGFVCKCY G33R 67 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGKFVCKCY G34K 68 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGRFVCKCY G34R 69 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGLVCKCY F35L 70 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFLCKCY V36L 71 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFMCKCY V36M 72 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFTCKCY V36T 73 3
GFGCKGPWDEDDMQCHNHCKSIKGYRGGYCAKGGFVCKCY N5K + K26R 74 3
GFGCKGPWDEGDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5K + D11G 75 3
GFGCSGPWDEDDMRCHNHCKAIRGYKGGYCAKGGFVCKCY N5S + Q14R + S21A + 76 3
K23R GFGCSGPWDEDDMRCHSHCKSIRGYKGGYCAKGGFVCKCY N5S + Q14R + N17S +
77 3 K23R GFGCNGPRDEDDRQCHNHCKSIKGYKGGYCAKGGFVCKCY W8R + M13R 78 3
GFGCNGPWGEDDMRCHNHCKSIRGYKGGYCAKGGFVCKCY D9G + Q14R + K23R 79 3
GFGCNGPWDGDDMRCHNHCKSIKGYKGGYCAKGGFVCKCY E10G + Q14R 80 3
GFGCNGPWDEGDMQCHNHCKSIKGYKGGYCARGGFVCKCY D11G + K32R 81 3
GFGCNGPWDEGDMQCHSHCKSIKGYKGGYCAKGGFVCKCY D11G + N17S 82 3
GFGCNGPWDEGDMQCHNHCKSVKGYKGGYCAKGGFVCKCY D11G + I22V 83 3
GFGCNGPWDENDMQCHNHCKSIKGYKGGYCAKGGFICKCY D11N + V36I 84 3
GFGCNGPWDERDIQCHNHCKSIKGYKGGYCAKGGFVCKCY D11R + M13I 85 3
GFGCNGPWDEDDMVCHNHCKSIKGYRGGYCAKGGFVCKCY Q14V + K26R 86 3
GFGCNGPWDEDDMRCHNHCKSIKGYRGGYCAKGGFVCRCY Q14R + K26R + K38R 87 3
GLGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY F2L 118 2
GWGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY F2W 119 2
GIGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY F2I 120 2
GFGCLGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5L 121 2
GFGCMGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5M 122 2
GFGCNRPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY G6R 123 2
GFGCNAPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY G6A 124 2
GFGCNKPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY G6K 125 2
GFGCNGAWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY P7A 126 2
GFGCNGLWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY P7L 127 2
GFGCNGVWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY P7V 128 2
GFGCNGPWCEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9C 129 2
GFGCNGPWIEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9I 130 2
GFGCNGPWREDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9R 131 2
GFGCNGPWWEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D9W 132 2
GFGCNGPWDADDMQCHNHCKSIKGYKGGYCAKGGFVCKCY E10A 133 2
GFGCNGPWDLDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY E10L 134 2
GFGCNGPWDCDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY E10C 135 2
GFGCNGPWDQDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY E10Q 136 2
GFGCNGPWDEADMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11A 137 2
GFGCNGPWDECDMQCHNHCKSIKGYKGGYCAKGGFVCKCY D11C 138 2
GFGCNGPWDEDPMQCHNHCKSIKGYKGGYCAKGGFVCKCY D12P 139 2
GFGCNGPWDEDDAQCHNHCKSIKGYKGGYCAKGGFVCKCY M13A 140 2
GFGCNGPWDEDDFQCHNHCKSIKGYKGGYCAKGGFVCKCY M13F 141 2
GFGCNGPWDEDDGQCHNHCKSIKGYKGGYCAKGGFVCKCY M13G 142 2
GFGCNGPWDEDDLQCHNHCKSIKGYKGGYCAKGGFVCKCY M13L 143 2
GFGCNGPWDEDDTQCHNHCKSIKGYKGGYCAKGGFVCKCY M13T 144 2
GFGCNGPWDEDDYQCHNHCKSIKGYKGGYCAKGGFVCKCY M13Y 145 2
GFGCNGPWDEDDMACHNHCKSIKGYKGGYCAKGGFVCKCY Q14A 146 2
GFGCNGPWDEDDMCCHNHCKSIKGYKGGYCAKGGFVCKCY Q14C 147 2
GFGCNGPWDEDDMICHNHCKSIKGYKGGYCAKGGFVCKCY Q14I 148 2
GFGCNGPWDEDDMKCHNHCKSIKGYKGGYCAKGGFVCKCY Q14K 149 2
GFGCNGPWDEDDMMCHNHCKSIKGYKGGYCAKGGFVCKCY Q14M 150 2
GFGCNGPWDEDDMPCHNHCKSIKGYKGGYCAKGGFVCKCY Q14P 151 2
GFGCNGPWDEDDMTCHNHCKSIKGYKGGYCAKGGFVCKCY Q14T 152 2
GFGCNGPWDEDDMVCHNHCKSIKGYKGGYCAKGGFVCKCY Q14V 153 2
GFGCNGPWDEDDMWCHNHCKSIKGYKGGYCAKGGFVCKCY Q14W 154 2
GFGCNGPWDEDDMQCHNACKSIKGYKGGYCAKGGFVCKCY H18A 155 2
GFGCNGPWDEDDMQCHNFCKSIKGYKGGYCAKGGFVCKCY H18F 156 2
GFGCNGPWDEDDMQCHNQCKSIKGYKGGYCAKGGFVCKCY H18Q 157 2
GFGCNGPWDEDDMQCHNTCKSIKGYKGGYCAKGGFVCKCY H18T 158 2
GFGCNGPWDEDDMQCHNVCKSIKGYKGGYCAKGGFVCKCY H18V 159 2
GFGCNGPWDEDDMQCHNHCQSIKGYKGGYCAKGGFVCKCY K20Q 160 2
GFGCNGPWDEDDMQCHNHCKSMKGYKGGYCAKGGFVCKCY I22M 161 2
GFGCNGPWDEDDMQCHNHCKSTKGYKGGYCAKGGFVCKCY I22T 162 2
GFGCNGPWDEDDMQCHNHCKSWKGYKGGYCAKGGFVCKCY I22W 163 2
GFGCNGPWDEDDMQCHNHCKSIKAYKGGYCAKGGFVCKCY G24A 164 2
GFGCNGPWDEDDMQCHNHCKSIKPYKGGYCAKGGFVCKCY G24P 165 2
GFGCNGPWDEDDMQCHNHCKSIKFYKGGYCAKGGFVCKCY G24F 166 2
GFGCNGPWDEDDMQCHNHCKSIKIYKGGYCAKGGFVCKCY G24I 167 2
GFGCNGPWDEDDMQCHNHCKSIKQYKGGYCAKGGFVCKCY G24Q 168 2
GFGCNGPWDEDDMQCHNHCKSIKRYKGGYCAKGGFVCKCY G24R 169 2
GFGCNGPWDEDDMQCHNHCKSIKSYKGGYCAKGGFVCKCY G24S 170 2
GFGCNGPWDEDDMQCHNHCKSIKTYKGGYCAKGGFVCKCY G24T 171 2
GFGCNGPWDEDDMQCHNHCKSIKYYKGGYCAKGGFVCKCY G24Y 172 2
GFGCNGPWDEDDMQCHNHCKSIKGHKGGYCAKGGFVCKCY Y25H 173 2
GFGCNGPWDEDDMQCHNHCKSIKGKKGGYCAKGGFVCKCY Y25K 174 2
GFGCNGPWDEDDMQCHNHCKSIKGLKGGYCAKGGFVCKCY Y25L 175 2
GFGCNGPWDEDDMQCHNHCKSIKGMKGGYCAKGGFVCKCY Y25M 176 2
GFGCNGPWDEDDMQCHNHCKSIKGNKGGYCAKGGFVCKCY Y25N 177 2
GFGCNGPWDEDDMQCHNHCKSIKGQKGGYCAKGGFVCKCY Y25Q 178 2
GFGCNGPWDEDDMQCHNHCKSIKGSKGGYCAKGGFVCKCY Y25S 179 2
GFGCNGPWDEDDMQCHNHCKSIKGVKGGYCAKGGFVCKCY Y25V 180 2
GFGCNGPWDEDDMQCHNHCKSIKGYFGGYCAKGGFVCKCY K26F 181 2
GFGCNGPWDEDDMQCHNHCKSIKGYHGGYCAKGGFVCKCY K26H 182 2
GFGCNGPWDEDDMQCHNHCKSIKGYTGGYCAKGGFVCKCY K26T 183 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGACAKGGFVCKCY Y29A 184 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGHCAKGGFVCKCY Y29H 185 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGLCAKGGFVCKCY Y29L 186 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGMCAKGGFVCKCY Y29M 187 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGSCAKGGFVCKCY Y29S 188 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCEKGGFVCKCY A31E 189 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCHKGGFVCKCY A31H 190 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCIKGGFVCKCY A31I 191 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCRKGGFVCKCY A31R 192 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCSKGGFVCKCY A31S 193 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCVKGGFVCKCY A31V 194 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCARGGFVCKCY K32R 195 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCATGGFVCKCY K32T 196 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKEGFVCKCY G33E 197 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKNGFVCKCY G33N 198 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKSGFVCKCY G33S 199 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKTGFVCKCY G33T 200 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGHFVCKCY G34H 201 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGWFVCKCY G34W 202 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGAVCKCY F35A 203 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGHVCKCY F35H 204 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGIVCKCY F35I 205 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGMVCKCY F35M 206 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGVVCKCY F35V 207 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGWVCKCY F35W 208 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFICKCY V36I 209 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFKCKCY V36K 210 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFQCKCY V36Q 211 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFRCKCY V36R 212 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCHCY K38H 213 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCNCY K38N 214 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCRCY K38R 215 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCI Y40I 216 2
GFGCNGPWLEDDMQCHNHCKSIKGYNGGYCAKGGFVCKCY D9L + K26N 217 2
GFGCNGPWDELDIQCHNHCKSIKGYKGGYCAKGGFVCKCY D11L + M13I 218 2
GFGCNGPWDEDDRQCHNHCKSIKGYKGGFCAKGGFVCKCY M13R + Y29F 219 2
GFGCNGPWDEDDMRCHNHCKSIRGYRGGYCAKGGFVCKCY Q14R + K23R + K26R 220 2
GFGCNGPWDEDDMRCHNHCRSIKGYKGGYCAKGGFVCKCY Q14R + K20R 221 2
GFGCNGPWDEDDMSCHNHCKSIKGYKGGYCAKGGFVCRCY Q14S + K38R 222 2
GFGCNGPWDEDDMQCHSHCKSIRGYKGGYCAKGGFVCKCY N17S + K23R 223 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGFCARGGFVCKCY Y29F + K32R 224 2
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCARGKFVCKCY K32R + G34K 225 2
Example 3
Identification of Antimicrobial Peptides with Improved
Antimicrobial Activity
[0287] A range of antimicrobial polypeptides, which are variants of
SEQ ID NO: 1, were tested in the TAPS assay. TAPS may be used to
identify new or improved genes encoding peptides that can kill or
inhibit the growth of target cells (see PCT application WO
2004/033715). The TAPS assay, an acronym for Trans Acting Peptide
System, is based on having a sensitive host producing a peptide
followed by screening for its activity in trans against an
indicator strain. The advantage of this system is based on that the
antimicrobial peptide is expressed in the Gram-negative bacterium
E. coli and its antimicrobial activity can be monitored on
different microbes including Gram-negatives, positives or fungi.
Additionally, TAPS offers the possibility to produce correctly
folded AMPs containing disulfide bonds in the host cells, thereby
retaining their antimicrobial activities.
[0288] The TAPS approach requires first, that expression of the
peptide is under control of an inducible promoter with tight
regulation, because the host cells are sensitive to the peptide
when producing it. Secondly, the produced peptide has to be
released to the media so that it can interact with the target
organism.
[0289] The TAPS screening can be carried out either in solid or
liquid media. On solid media, a plasmid library is initially
introduced into E. coli host cells. It is important that the
transformants are cultivated on the surface of a cellulose acetate
filter placed on LB growth medium without inducer (arabinose) to
avoid expression of the antimicrobial peptide and hence growth
inhibition. In the next step, the filter containing the colonies is
transferred to LB growth medium containing inducer (0.1% arabinose)
to permit peptide synthesis. Subsequently, the target strain, for
example S. carnosus, is overlaid onto the plate and allowed to grow
for 12-16 hours at 37.degree. C. Finally, visual inspection of the
host cells capable of reducing the proliferation of the target
cells is performed and the nucleotide sequence encoding for the
antimicrobial peptide is recovered from the host cells. DNA
sequence analysis of the variants is obtained to elucidate the
nature of the peptide.
[0290] As mentioned above, the TAPS screening can also be performed
using liquid medium. This procedure requires the use of robotics to
analyze large number of clones. In this system, the host E. coli
origami cells are transformed with the plasmid library and plated
out on LB medium+0.2% glucose+ampicillin (200 mg/L). Independent
colonies are then inoculated into 96 or 384-well plates containing
200 micro-L of TB medium+ampicillin (200 mg/L) and cultured
overnight at 37.degree. C. These cultures are then replicated
robotically and grown to exponential phase until inducer (0.1%
arabinose) is added to trigger peptide synthesis. The next step
consists in hydrolyzing the cells such that the peptide is released
to the media by hot acid hydrolysis. This treatment consists on
adding 1 M sodium Phosphate buffer pH 2.3 to obtain a final pH
approximately of 2.3 and incubating the cultures overnight at
80.degree. C. Next day, a 25 micro-L aliquot of the hydrolyzed
cultures is used to perform an activity test against the desired
target organisms. The activity test performed was a Radial
Diffusion Assay (RDA) where an aliquot of the hydrolyzed cultures
was added to the agarose media inoculated with the target strain,
S. carnosus. RDAs obtained from the screening plates containing
clearing zones corresponding to clones exhibiting antimicrobial
activity were easily identified. Measurements of the diameter of
the clearing zones were performed to quantify the potency of the
antimicrobial activity of the peptides.
[0291] The antimicrobial activity (against Staphylococcus carnosus)
of the tested plectasin variants, which was measured using the TAPS
assay, is shown in table 3. The antimicrobial activity corresponds
to the clearing zone size and has been classified as
4>3>2>1; whereas 4 is better than 1, and wildtype activity
corresponds to 1.
TABLE-US-00006 TABLE 3 Antimicrobial activity data from the TAPS
assay SEQ ID Sequence Mutation(s) NO: Activity control 0
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY wildtype 1 1
GFGCKGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5K 88 2
GFGCYGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY N5Y 89 2
GFGCNGPWDEDDKQCHNHCKSIKGYKGGYCAKGGFVCKCY M13K 90 3
GFGCNGPWDEDDMLCHNHCKSIKGYKGGYCAKGGFVCKCY Q14L 91 4
GFGCNGPWDEDDMRCHNHCKSIKGYKGGYCAKGGFVCKCY Q14R 92 4
GFGCNGPWDEDDMQCHHHCKSIKGYKGGYCAKGGFVCKCY N17H 93 4
GFGCNGPWDEDDMQCHIHCKSIKGYKGGYCAKGGFVCKCY N17I 94 2
GFGCNGPWDEDDMQCHRHCKSIKGYKGGYCAKGGFVCKCY N17R 95 2
GFGCNGPWDEDDMQCHYHCKSIKGYKGGYCAKGGFVCKCY N17Y 96 3
GFGCNGPWDEDDMQCHNHCRSIKGYKGGYCAKGGFVCKCY K20R 97 2
GFGCNGPWDEDDMQCHNHCKSIKGYRGGYCAKGGFVCKCY K26R 98 3
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCYR Y40YR 99 2
GFGCYGPWDEDDMLCHNHCKSIKGYKGGYCAKGGFVCKCY N5Y + Q14L 100 2
GFGCNGPWDEGDMQCHNHCKSIKGYRGGYCAKGGFVCKCY D11G + K26R 101 2
GFGCNGPWDEGDKQCHNHCKSIKGYRGGYCAKGGFVCKCY D11G + M13K + K26R 102 3
GFGCNGPWDEDDKQCHRHCKSIKGYKGGYCAKGGFVCKCYR M13K + N17R + Y40YR 103 3
GFGCNGPWDEDDMQCHRHCKSIKGYKGGYCAKGGFVCKCYR N17R + Y40YR 104 3
GFGCNGPWDENDMQCHNHCKFIKGYKGGYCAKGGFVCKCY D11N + S21F 105 4
GFGCNGPWDEDDKQCHRHCKSIKGYKGGYCAKGGFVCKCY M13K + N17R 106 3
GFGCNGPWDEDDKQCHNHCKSIKGYKGGYCAKGGFVCKCYR M13K + Y40YR 107 4
GFGCNGPWDEDDKQCHNHCKSIKGYRGGYCAKGGFVCKCY M13K + K26R 108 4
GFGCNGPWDEDDKQCHNHCKSIKGYKGGYCAKGGFVCRCY M13K + K38R 109 4
GFGCNGPWDEGDMQCHNHCKSIKGYRGGYCAKGGFVCKCYR D11G + K26R + Y40YR 110 4
GFGCNGPWDEGDKQCHNHCKSIKGYRGGYCAKGGFVCKCYR D11G + M13K + K26R + 111
4 Y40YR GFGCNGPWDEDDMRCHNHCKSIKGYKGGYCAKGGFVCKCYR Q14R + Y40YR 112
3 GFGCNGPWDEDDMRCHNHCKSIKGYKGGYCAKGGFICKCY Q14R + V36I 113 4
GFGCNGPWDEDDMRCHNHCKSIKGYRGGYCAKGGFVCKCY Q14R + K26R 114 4
GFGCNGPWDEDDMQCHNHCKFIKGYKGGYCTKGGFVCKCY S21F + A31T 115 2
Example 4
Evaluation of Antimicrobial Activity
[0292] Plectasin wild-type (SEQ ID NO: 1) and five variants of
Plectasin, which showed improved activity in the TAPS method and/or
in the yeast system, were expressed and purified. The Minimal
Inhibitory Concentration (MIC, micrograms/mL) was determined to
test for their antimicrobial activity following the NCCLS
guidelines (Clinical and Laboratory Standards Institute; formerly
known as National Committee for Clinical and Laboratory
Standards).
[0293] The results showed that all plectasin variants had improved
activity, compared to wildtype plectasin, against Staphylococcus
aureus, Micrococcus luteus and Bacillus subtilis.
TABLE-US-00007 TABLE 4 MIC values; all values are micrograms/mL SEQ
MIC against MIC against MIC against Mutation(s) ID NO: S. aureus M.
luteus B. subtilis Wildtype 1 8 32 1 M13K + Y40YR 107 2 2 0.25 D11G
+ K26R 101 2 8 0.50 Q14K + K26R 116 2 1 0.13 D11G + K26R + 110 0.50
0.25 0.06 Y40YR D11G + M13K + 111 0.50 0.50 <0.03 K26R +
Y40YR
Example 5
Evaluation of Antimicrobial Activity
[0294] Plectasin wild-type (SEQ ID NO: 1) and several variants of
Plectasin were expressed and purified. The Minimal Inhibitory
Concentration (MIC) was determined to test for their antimicrobial
activity following the NCCLS guidelines from CLSI (Clinical and
Laboratory Standards Institute; formerly known as National
Committee for Clinical and Laboratory Standards).
[0295] The peptides were tested against the following strains:
[0296] A: Staphylococcus aureus, ATCC 29213; [0297] B:
Staphylococcus aureus, ATCC 25923; [0298] C: Staphylococcus aureus,
ATCC 29737; [0299] D: Staphylococcus aureus, MRSA ST5 (2001),
multi-resistant clinical human isolate from Statens Serum Institut,
Denmark; [0300] E: Staphylococcus aureus, MRSA ST80 (2003),
multi-resistant clinical human isolate from Statens Serum Institut,
Denmark.
[0301] All MIC values represent an average of two independent
experiments; and all results in Table 5 are expressed relative to
wildtype Plectasin:
0: >100% of Plectasin wildtype MIC; 1: 80-100% of Plectasin
wildtype MIC; 2: 50-80% of Plectasin wildtype MIC; 3: <50% of
Plectasin wildtype MIC.
TABLE-US-00008 TABLE 5 Relative MIC values; nd = not determined SEQ
ID Mutation(s) NO: A B C D E wildtype 1 1 1 1 1 1 (8 .mu.g/mL) (22
.mu.g/mL) (4 .mu.g/mL) (15 .mu.g/mL) (22 .mu.g/mL) N5R + M13Y +
N17R 226 3 3 3 3 3 D9S + Q14K + V36L 227 3 3 3 3 3 N5S + M13W +
N17R 228 3 nd 3 3 3 Q14R + K26R + K38R 87 3 3 3 3 3 D9G + Q14R +
K23R 79 3 3 3 3 3 M13G + N17R + G33A 229 3 3 3 3 3 N5S + D9S + M13L
+ Q14R + 230 3 3 3 3 3 N17V + A31S D9S + Q14L + K26R 231 3 3 3 3 3
N5S + D9A + K26R 232 3 3 3 3 3 Q14R + K20R 221 3 3 3 3 3 N5G + M13L
233 2 3 3 3 3 D9A + K38R 234 3 3 3 3 3 D11G + K32R 81 1 3 3 2 3
Q14F 46 1 nd 3 2 3 N5R + M13V 235 3 nd 3 2 3 N5G + M13Y + N17K 236
3 3 3 3 2 Q14K + K26R 116 3 2 3 3 3 M13F + Q14K + K26R 237 3 nd 3 3
3 M13K + K38R 109 1 3 3 2 3 N5A + D9S + M13L + N17T 238 0 3 3 2 3
N17Y 96 1 3 3 2 2 M13T + Q14K + K26R 239 1 3 3 2 0 D9S 24 2 3 3 2
nd N17R 95 nd 3 3 3 2 Q14R 92 2 3 3 3 0 N5S + Q14R + N17S + K23R 77
1 3 3 3 nd N5R 3 1 2 2 2 2 D11G + K26R + Y40YR 110 3 3 3 3 nd N5S +
Q14R + S21A + K23R 76 1 3 2 2 0 M13L + Q14K + K26R 240 3 nd 3 3 nd
D9N + M13L + Q14R 241 3 3 3 3 3 D9A + Q14H + K26R + V36L 242 3 3 3
3 3 Q14R + K23R + K26R 220 3 3 3 3 3 N5S + D9S + M13V + N17R 243 3
3 3 3 3 N5G + D9S + M13L + 244 3 3 3 3 3 N17Q + A31T M13V + N17T
245 nd 3 3 3 3 D9S + M13L + Q14H 246 3 3 3 3 3 D9S + Q14L 247 3 nd
3 3 3 D9N + Q14H + K38R 248 3 3 3 3 2 M13Y + Q14K + K26R 249 2 3 3
3 3 D11N 40 2 3 2 2 3 N5S + D9S 250 0 3 3 2 3 G24R 169 1 2 nd 2 2
N5G + Q14K 251 2 2 3 0 2 N5K + K26R 74 1 2 3 2 2
Example 6
Evaluation of Antimicrobial Activity
[0302] A large number of antimicrobial peptides of the invention
were tested against a panel of 6 different strains of
Staphylococcus aureus listed below:
Staphylococcus aureus, ATCC29213, MSSA, NCCLS reference strain;
Staphylococcus aureus, ATCC25923, MSSA, NCCLS reference strain;
Staphylococcus aureus, ATCC29737, MSSA; Staphylococcus aureus,
E33235, MSSA; Staphylococcus aureus, 698-01, MRSA ST5, Str, Kan,
Oxa; Staphylococcus aureus, 566-03, MRSA ST80, Oxa, Tet, Fus,
Kan.
[0303] S. aureus E33235, S. aureus 698-01 and S. aureus 566-03 are
available from Statens Serum Institut, Denmark.
[0304] The staphylococci were exposed to the following peptide
concentrations: 32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.13; 0.6; and 0.03
microgram/mL. All peptides were purified, HPLC quantified and the
concentrated peptides (>160 micrograms/mL) were diluted to 160
micrograms/mL in peptide dilution buffer (0.1% BSA, 0.01% Acetic
Acid).
[0305] The MIC determination was done essentially as described by
NCCLS/CLSI guidelines using caMHB. The MICs were read after 18-24
hours of 37.degree. C. incubations and recorded along with the CFU
in the table below.
[0306] A total number of 95 antimicrobial peptides of the invention
were evaluated in duplicate against the 6 bacterial strains
described above.
[0307] In the majority of the double determinations (93%), the MIC
varied <2 fold. An average MIC is tabulated below. If the MIC
was above 32 micrograms/mL, a value of 64 was used to calculate the
average.
TABLE-US-00009 TABLE 6 Average MIC values Average MIC Mutation(s)
SEQ ID NO: (micrograms/mL) N5R + M13Y + N17R 226 1 D9N + M13L +
Q14R 241 1 D9S + Q14K + V36L 227 1 D9A + Q14H + K26R + V36L 242 1
D9G + Q14R + K23R 79 2 Q14R + K23R + K26R 220 2 M13G + N17R + G33A
229 2 N5S + D9S + M13V + N17R 243 2 N5S + M13W + N17R 228 2 Q14R +
K26R + K38R 87 2 N5S + D9S + M13L + Q14R + 230 2 N17V + A31S D9S +
Q14L + K26R 231 3 N5S + D9A + K26R 232 3 N5G + D9S + M13L + N17Q +
A31T 244 3 M13V + N17T 245 3 D9S + M13L + Q14H 246 3 D9S + Q14L 247
3 Q14R + K20R 221 3 N5G + M13L 233 4 D9A + K38R 234 4 D9N + Q14H +
K38R 248 4 N17R 95 4 M13R + Q14K + K26R 252 5 M13Y + Q14K + K26R
249 5 D11G + K32R 81 5 Q14F 46 5 N5R + M13V 235 5 Q14R 92 5 N5G +
M13Y + N17K 236 5 Q14R + K26R 114 5 M13F + Q14K + K26R 237 6 M13K +
K38R 109 6 D11N 40 6 N5S + D9S 250 6 Q14K + K26R 116 6 N5A + D9S +
M13L + N17T 238 6 N17Y 96 6 N17K + K32R 253 7 M13T + Q14K + K26R
239 7 D9K 14 7 D9S 24 7 M13K + N17R + Y40YR 103 8 N5S + Q14R + N17S
+ K23R 77 8 N5R 3 8 G24R 169 8 D11G + K26R + Y40YR 110 9 N5S + Q14R
+ S21A + K23R 76 9 N5G + M13W 254 9 N5G + Q14K 251 9 M13A + Q14K +
K26R 255 9 M13L + Q14K + K26R 240 9 N5K + K26R 74 9 A31N 61 9 M13R
43 9 N5K + M13L 256 9 M13K + K26R 108 10 N5G + M13W + N17S 257 10
N17K 258 11 S21N 259 11 D11G + K26R 101 11 K26C + Y40YRCG 260 11
Q14V + K26R 86 12 M13K + Y40YR 107 12 M13K 90 12 N5H + M13E + N17E
261 12 N5K 88 12 N17F 262 12 D11G + M13K + K26R + Y40YR 111 12 Q14L
91 13 Wild-type 1 14 N17I 94 14 N17S 263 14 N17S + K23R 223 14 N5G
+ D9A + Q14S + K23T + A31T 264 14 N17A 265 15 K26R 98 15 M13S +
Q14K + K26R 266 15 D11G + N17I 267 16 N5S + M13V + N17T 268 16 N5D
+ D9S + Q14R 269 16 D11G + I22V 83 17 S21A 270 17 N17R + Y25R 271
18 N5S + M13V + N17A 272 18 Q14G 47 18 D9G 13 21 N17T 273 21 Q14S
49 21 N5G 6 24 N5S 7 24 M13S 44 24 S21V 274 26 M13T 144 26 V36L 71
26 N5A 8 29 D9V 25 30
Example 7
Using the HMM Files from the PFAM Database to Identify a
Defensin
[0308] Sequence analysis using hidden markov model profiles (HMM
profiles) may be carried out either online on the Internet or
locally on a computer using the well-known HMMER freely available
software package. The current version is HMMER 2.3.2 from October
2003.
[0309] The HMM profiles may be obtained from the well-known PFAM
database. The current version is PFAM 16.0 from November 2004. Both
HMMER and PFAM are available for all computer platforms from, e.g.,
Washington University in St. Louis (USA), School of Medicine
(http://pfam.wustl.edu and http://hmmer.wustl.edu).
[0310] If a query amino acid sequence, or a fragment thereof,
belongs to one of the following five PFAM families, the amino acid
sequence is a defensin according to the present invention:
[0311] Defensin_beta or "Beta Defensin", accession number:
PF00711;
[0312] Defensin_propep or "Defensin propeptide", accession number:
PF00879;
[0313] Defensin.sub.--1 or "Mammalian defensin", accession number:
PF00323;
[0314] Defensin.sub.--2 or "Arthropod defensin", accession number:
PF01097;
[0315] Gamma-thionin or "Gamma-thionins family", accession number:
PF00304.
[0316] An amino acid sequence belongs to a PFAM family, according
to the present invention, if it generates an E-value which is
greater than 0.1, and a score which is larger or equal to zero,
when the PFAM database is used online, or when the hmmpfam program
(from the HMMER software package) is used locally.
[0317] When the sequence analysis is carried out locally using the
hmmpfam program, it is necessary to obtain (download) the HMM
profiles from the PFAM database. Two profiles exist for each
family; xxx_ls.hmm for glocal searches, and xxx_fs.hmm for local
searches ("xxx" is the name of the family). That makes a total of
ten profiles for the five families mentioned above.
[0318] These ten profiles may be used individually, or joined
(appended) into a single profile (using a text editor--the profiles
are ASCII files) that could be named, e.g., defensin.hmm. A query
amino acid sequence can then be evaluated by using the following
command line: [0319] hmmpfam -E 0.1 defensin.hmm sequence_file
[0320] wherein "sequence_file" is a file with the query amino acid
sequence in any of the formats recognized by the HMMER software
package.
[0321] If the score is larger or equal to zero (0.0), and the
E-value is greater than 0.1, the query amino acid sequence is a
defensin according to the present invention.
[0322] The PFAM database is further described in Bateman et al.
(2004) "The Pfam Protein Families Database", Nucleic Acids
Research, Vol. 32 (Database Issue) pp. D138-D141.
Sequence CWU 1
1
274140PRTPseudoplectania nigrella 1Gly Phe Gly Cys Asn Gly Pro Trp
Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys
Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys
Cys Tyr 35 40243PRTArtificial sequenceSynthetic construct 2Gly Xaa
Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys His1 5 10 15Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Gly Xaa Cys Xaa Xaa 20 25
30Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 35 40340PRTArtificial
sequenceSynthetic construct 3Gly Phe Gly Cys Arg Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 40440PRTArtificial sequenceSynthetic construct 4Gly Phe Gly
Cys Gln Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His
Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly
Gly Phe Val Cys Lys Cys Tyr 35 40540PRTArtificial sequenceSynthetic
construct 5Gly Phe Gly Cys Val Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
40640PRTArtificial sequenceSynthetic construct 6Gly Phe Gly Cys Gly
Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys
Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe
Val Cys Lys Cys Tyr 35 40740PRTArtificial sequenceSynthetic
construct 7Gly Phe Gly Cys Ser Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
40840PRTArtificial sequenceSynthetic construct 8Gly Phe Gly Cys Ala
Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys
Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe
Val Cys Lys Cys Tyr 35 40940PRTArtificial sequenceSynthetic
construct 9Gly Phe Gly Cys Asn Gly Lys Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
401040PRTArtificial sequenceSynthetic construct 10Gly Phe Gly Cys
Asn Gly Arg Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 401140PRTArtificial sequenceSynthetic
construct 11Gly Phe Gly Cys Asn Gly Pro Arg Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
401240PRTArtificial sequenceSynthetic construct 12Gly Phe Gly Cys
Asn Gly Pro Trp Ala Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 401340PRTArtificial sequenceSynthetic
construct 13Gly Phe Gly Cys Asn Gly Pro Trp Gly Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
401440PRTArtificial sequenceSynthetic construct 14Gly Phe Gly Cys
Asn Gly Pro Trp Lys Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 401540PRTArtificial sequenceSynthetic
construct 15Gly Phe Gly Cys Asn Gly Pro Trp Leu Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
401640PRTArtificial sequenceSynthetic construct 16Gly Phe Gly Cys
Asn Gly Pro Trp Thr Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 401740PRTArtificial sequenceSynthetic
construct 17Gly Phe Gly Cys Asn Gly Pro Trp Tyr Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
401840PRTArtificial sequenceSynthetic construct 18Gly Phe Gly Cys
Asn Gly Pro Trp Phe Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 401940PRTArtificial sequenceSynthetic
construct 19Gly Phe Gly Cys Asn Gly Pro Trp His Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
402040PRTArtificial sequenceSynthetic construct 20Gly Phe Gly Cys
Asn Gly Pro Trp Met Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 402140PRTArtificial sequenceSynthetic
construct 21Gly Phe Gly Cys Asn Gly Pro Trp Asn Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
402240PRTArtificial sequenceSynthetic construct 22Gly Phe Gly Cys
Asn Gly Pro Trp Pro Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 402340PRTArtificial sequenceSynthetic
construct 23Gly Phe Gly Cys Asn Gly Pro Trp Gln Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
402440PRTArtificial sequenceSynthetic construct 24Gly Phe Gly Cys
Asn Gly Pro Trp Ser Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 402540PRTArtificial sequenceSynthetic
construct 25Gly Phe Gly Cys Asn Gly Pro Trp Val Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
402640PRTArtificial sequenceSynthetic construct 26Gly Phe Gly Cys
Asn Gly Pro Trp Asp Gly Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 402740PRTArtificial sequenceSynthetic
construct 27Gly Phe Gly Cys Asn Gly Pro Trp Asp Ser Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
402840PRTArtificial sequenceSynthetic construct 28Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Phe Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 402940PRTArtificial sequenceSynthetic
construct 29Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Gly Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
403040PRTArtificial sequenceSynthetic construct 30Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu His Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 403140PRTArtificial sequenceSynthetic
construct 31Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Lys Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
403240PRTArtificial sequenceSynthetic construct 32Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Leu Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 403340PRTArtificial sequenceSynthetic
construct 33Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Pro Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
403440PRTArtificial sequenceSynthetic construct 34Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Ser Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 403540PRTArtificial sequenceSynthetic
construct 35Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Thr Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
403640PRTArtificial sequenceSynthetic construct 36Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Val Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 403740PRTArtificial sequenceSynthetic
construct 37Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Trp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
403840PRTArtificial sequenceSynthetic construct 38Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Ile Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 403940PRTArtificial sequenceSynthetic
construct 39Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Met Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
404040PRTArtificial sequenceSynthetic construct 40Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asn Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 404140PRTArtificial sequenceSynthetic
construct 41Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Arg Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
404240PRTArtificial sequenceSynthetic construct 42Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Tyr Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 404340PRTArtificial sequenceSynthetic
construct 43Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Arg Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
404440PRTArtificial sequenceSynthetic construct 44Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Ser Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 404540PRTArtificial sequenceSynthetic
construct 45Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Val Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
404640PRTArtificial sequenceSynthetic construct 46Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Phe Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 404740PRTArtificial sequenceSynthetic
construct 47Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gly
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
404840PRTArtificial sequenceSynthetic construct 48Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met His Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 404940PRTArtificial sequenceSynthetic
construct 49Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Ser
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
405040PRTArtificial sequenceSynthetic construct 50Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Tyr Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 405140PRTArtificial sequenceSynthetic
construct 51Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn Leu Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
405240PRTArtificial sequenceSynthetic construct 52Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Leu Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 405340PRTArtificial sequenceSynthetic
construct 53Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Val Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
405440PRTArtificial sequenceSynthetic construct 54Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys His Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 405540PRTArtificial sequenceSynthetic
construct 55Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Lys Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
405640PRTArtificial sequenceSynthetic construct 56Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Asn Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 405740PRTArtificial sequenceSynthetic
construct 57Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Phe
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
405840PRTArtificial sequenceSynthetic construct 58Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Arg Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 405940PRTArtificial sequenceSynthetic
construct 59Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Trp
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
406040PRTArtificial sequenceSynthetic construct 60Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Lys Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 406140PRTArtificial sequenceSynthetic
construct 61Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Asn Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
406240PRTArtificial sequenceSynthetic construct 62Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Gln Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 406340PRTArtificial sequenceSynthetic
construct 63Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Thr Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
406440PRTArtificial sequenceSynthetic construct 64Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Tyr Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 406540PRTArtificial sequenceSynthetic
construct 65Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Lys Gly Phe Val Cys Lys Cys Tyr 35
406640PRTArtificial sequenceSynthetic construct 66Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gln Gly
Phe Val Cys Lys Cys Tyr 35 406740PRTArtificial sequenceSynthetic
construct 67Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Arg Gly Phe Val Cys Lys Cys Tyr 35
406840PRTArtificial sequenceSynthetic construct 68Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Lys
Phe Val Cys Lys Cys Tyr 35 406940PRTArtificial sequenceSynthetic
construct 69Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Arg Phe Val Cys Lys Cys Tyr 35
407040PRTArtificial sequenceSynthetic construct 70Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Leu Val Cys Lys Cys Tyr 35 407140PRTArtificial sequenceSynthetic
construct 71Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Leu Cys Lys Cys Tyr 35
407240PRTArtificial sequenceSynthetic construct 72Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Met Cys Lys Cys Tyr 35 407340PRTArtificial sequenceSynthetic
construct 73Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Thr Cys Lys Cys Tyr 35
407440PRTArtificial sequenceSynthetic construct 74Gly Phe Gly Cys
Lys Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 407540PRTArtificial sequenceSynthetic
construct 75Gly Phe Gly Cys Lys Gly Pro Trp Asp Glu Gly Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
407640PRTArtificial sequenceSynthetic construct 76Gly Phe Gly Cys
Ser Gly Pro Trp Asp Glu Asp Asp Met Arg Cys His1 5 10 15Asn His Cys
Lys Ala Ile Arg Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 407740PRTArtificial sequenceSynthetic
construct 77Gly Phe Gly Cys Ser Gly Pro Trp Asp Glu Asp Asp Met Arg
Cys His1 5 10 15Ser His Cys Lys Ser Ile Arg Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
407840PRTArtificial sequenceSynthetic construct 78Gly Phe Gly Cys
Asn Gly Pro Arg Asp Glu Asp Asp Arg Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 407940PRTArtificial sequenceSynthetic
construct 79Gly Phe Gly Cys Asn Gly Pro Trp Gly Glu Asp Asp Met Arg
Cys His1 5 10 15Asn His Cys Lys Ser Ile Arg Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
408040PRTArtificial sequenceSynthetic construct 80Gly Phe Gly Cys
Asn Gly Pro Trp Asp Gly Asp Asp Met Arg Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 408140PRTArtificial sequenceSynthetic
construct 81Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Gly Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Arg 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
408240PRTArtificial sequenceSynthetic construct 82Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Gly Asp Met Gln Cys His1 5 10 15Ser His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 408340PRTArtificial sequenceSynthetic
construct 83Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Gly Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Val Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
408440PRTArtificial sequenceSynthetic construct 84Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asn Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Ile Cys Lys Cys Tyr 35 408540PRTArtificial sequenceSynthetic
construct 85Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Arg Asp Ile Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
408640PRTArtificial sequenceSynthetic construct 86Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Val Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 408740PRTArtificial sequenceSynthetic
construct 87Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Arg
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Arg Cys Tyr 35
408840PRTArtificial sequenceSynthetic construct 88Gly Phe Gly Cys
Lys Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 408940PRTArtificial sequenceSynthetic
construct 89Gly Phe Gly Cys Tyr Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
409040PRTArtificial sequenceSynthetic construct 90Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Lys Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 409140PRTArtificial sequenceSynthetic
construct 91Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Leu
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
409240PRTArtificial sequenceSynthetic construct 92Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Arg Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 409340PRTArtificial sequenceSynthetic
construct 93Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15His His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
409440PRTArtificial sequenceSynthetic construct 94Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Ile His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 409540PRTArtificial sequenceSynthetic
construct 95Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Arg His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
409640PRTArtificial sequenceSynthetic construct 96Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Tyr His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 409740PRTArtificial sequenceSynthetic
construct 97Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Arg Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
409840PRTArtificial sequenceSynthetic construct 98Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 409941PRTArtificial sequenceSynthetic
construct 99Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln
Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr Arg 35
4010040PRTArtificial sequenceSynthetic construct 100Gly Phe Gly Cys
Tyr Gly Pro Trp Asp Glu Asp Asp Met Leu Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4010140PRTArtificial sequenceSynthetic
construct 101Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Gly Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly
Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4010240PRTArtificial
sequenceSynthetic construct 102Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Gly Asp Lys Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4010341PRTArtificial sequenceSynthetic construct 103Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Lys Gln Cys His1 5 10 15Arg
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr Arg 35 4010441PRTArtificial
sequenceSynthetic construct 104Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Arg His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr Arg 35 4010540PRTArtificial sequenceSynthetic construct 105Gly
Phe Gly Cys Asn Gly Pro Trp Asp Glu Asn Asp Met Gln Cys His1 5 10
15Asn His Cys Lys Phe Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys
20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35 4010640PRTArtificial
sequenceSynthetic construct 106Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Lys Gln Cys His1 5 10 15Arg His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4010741PRTArtificial sequenceSynthetic construct 107Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Lys Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr Arg 35 4010840PRTArtificial
sequenceSynthetic construct 108Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Lys Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4010940PRTArtificial sequenceSynthetic construct 109Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Lys Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Arg Cys Tyr 35 4011041PRTArtificial
sequenceSynthetic antimicrobial polypeptide 110Gly Phe Gly Cys Asn
Gly Pro Trp Asp Glu Gly Asp Met Gln Cys His1 5 10 15Asn His Cys Lys
Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe
Val Cys Lys Cys Tyr Arg 35 4011141PRTArtificial sequenceSynthetic
construct 111Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Gly Asp Lys
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr Arg 35
4011241PRTArtificial sequenceSynthetic construct 112Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Arg Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr Arg 35 4011340PRTArtificial
sequenceSynthetic construct 113Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Arg Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Ile Cys Lys Cys
Tyr 35 4011440PRTArtificial sequenceSynthetic construct 114Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Arg Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4011540PRTArtificial
sequenceSynthetic construct 115Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Phe Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Thr Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4011640PRTArtificial sequenceSynthetic construct 116Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Lys Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4011740PRTArtificial
sequenceSynthetic construct 117Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Arg Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4011840PRTArtificial sequenceSynthetic construct 118Gly Leu
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4011940PRTArtificial
sequenceSynthetic construct 119Gly Trp Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4012040PRTArtificial sequenceSynthetic construct 120Gly Ile
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4012140PRTArtificial
sequenceSynthetic construct 121Gly Phe Gly Cys Leu Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4012240PRTArtificial sequenceSynthetic construct 122Gly Phe
Gly Cys Met Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4012340PRTArtificial
sequenceSynthetic construct 123Gly Phe Gly Cys Asn Arg Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4012440PRTArtificial sequenceSynthetic construct 124Gly Phe
Gly Cys Asn Ala Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4012540PRTArtificial
sequenceSynthetic construct 125Gly Phe Gly Cys Asn Lys Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4012640PRTArtificial sequenceSynthetic construct 126Gly Phe
Gly Cys Asn Gly Ala Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4012740PRTArtificial
sequenceSynthetic construct 127Gly Phe Gly Cys Asn Gly Leu Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4012840PRTArtificial sequenceSynthetic construct 128Gly Phe
Gly Cys Asn Gly Val Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4012940PRTArtificial
sequenceSynthetic construct 129Gly Phe Gly Cys Asn Gly Pro Trp Cys
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4013040PRTArtificial sequenceSynthetic construct 130Gly Phe
Gly Cys Asn Gly Pro Trp Ile Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4013140PRTArtificial
sequenceSynthetic construct 131Gly Phe Gly Cys Asn Gly Pro Trp Arg
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4013240PRTArtificial sequenceSynthetic construct 132Gly Phe
Gly Cys Asn Gly Pro Trp Trp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4013340PRTArtificial
sequenceSynthetic construct 133Gly Phe Gly Cys Asn Gly Pro Trp Asp
Ala Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4013440PRTArtificial sequenceSynthetic construct 134Gly Phe
Gly Cys Asn Gly Pro Trp Asp Leu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4013540PRTArtificial
sequenceSynthetic construct 135Gly Phe Gly Cys Asn Gly Pro Trp Asp
Cys Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4013640PRTArtificial sequenceSynthetic construct 136Gly Phe
Gly Cys Asn Gly Pro Trp Asp Gln Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4013740PRTArtificial
sequenceSynthetic construct 137Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Ala Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4013840PRTArtificial sequenceSynthetic construct 138Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Cys Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4013940PRTArtificial
sequenceSynthetic construct 139Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Pro Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4014040PRTArtificial sequenceSynthetic construct 140Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Ala Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4014140PRTArtificial
sequenceSynthetic construct 141Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Phe Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4014240PRTArtificial sequenceSynthetic construct 142Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Gly Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4014340PRTArtificial
sequenceSynthetic construct 143Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Leu Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4014440PRTArtificial sequenceSynthetic construct 144Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Thr Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4014540PRTArtificial
sequenceSynthetic construct 145Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Tyr Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4014640PRTArtificial sequenceSynthetic construct 146Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Ala Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4014740PRTArtificial
sequenceSynthetic construct 147Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Cys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4014840PRTArtificial sequenceSynthetic construct 148Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Ile Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4014940PRTArtificial
sequenceSynthetic construct 149Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4015040PRTArtificial sequenceSynthetic construct 150Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Met Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4015140PRTArtificial
sequenceSynthetic construct 151Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Pro Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35
4015240PRTArtificial sequenceSynthetic construct 152Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Thr Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4015340PRTArtificial sequenceSynthetic
construct 153Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Val Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4015440PRTArtificial sequenceSynthetic construct 154Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Trp Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4015540PRTArtificial sequenceSynthetic
construct 155Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn Ala Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4015640PRTArtificialSynthetic antimicrobial polypeptide 156Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
Phe Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4015740PRTArtificial
sequenceSynthetic construct 157Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn Gln Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4015840PRTArtificial sequenceSynthetic construct 158Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
Thr Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4015940PRTArtificial
sequenceSynthetic construct 159Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn Val Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4016040PRTArtificial sequenceSynthetic construct 160Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Gln Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4016140PRTArtificial
sequenceSynthetic construct 161Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Met Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4016240PRTArtificial sequenceSynthetic construct 162Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Thr Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4016340PRTArtificial
sequenceSynthetic construct 163Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Trp Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4016440PRTArtificial sequenceSynthetic construct 164Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Ala Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4016540PRTArtificial
sequenceSynthetic construct 165Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Pro
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4016640PRTArtificial sequenceSynthetic construct 166Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Phe Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4016740PRTArtificial
sequenceSynthetic construct 167Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Ile
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4016840PRTArtificial sequenceSynthetic construct 168Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gln Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4016940PRTArtificial
sequenceSynthetic construct 169Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Arg
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4017040PRTArtificial sequenceSynthetic construct 170Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Ser Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4017140PRTArtificial
sequenceSynthetic construct 171Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Thr
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4017240PRTArtificial sequenceSynthetic construct 172Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Tyr Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4017340PRTArtificial
sequenceSynthetic construct 173Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
His Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4017440PRTArtificial sequenceSynthetic construct 174Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Lys Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4017540PRTArtificial
sequenceSynthetic construct 175Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Leu Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4017640PRTArtificial sequenceSynthetic construct 176Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Met Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4017740PRTArtificial
sequenceSynthetic construct 177Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Asn Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4017840PRTArtificial sequenceSynthetic construct 178Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Gln Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4017940PRTArtificial
sequenceSynthetic construct 179Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Ser Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4018040PRTArtificial sequenceSynthetic construct 180Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Val Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4018140PRTArtificial
sequenceSynthetic construct 181Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Phe Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4018240PRTArtificial sequenceSynthetic construct 182Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr His Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4018340PRTArtificial
sequenceSynthetic construct 183Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Thr Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4018440PRTArtificial sequenceSynthetic construct 184Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Ala Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4018540PRTArtificial
sequenceSynthetic construct 185Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly His Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4018640PRTArtificial sequenceSynthetic construct 186Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Leu Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4018740PRTArtificial
sequenceSynthetic construct 187Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Met Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4018840PRTArtificial sequenceSynthetic construct 188Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Ser Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4018940PRTArtificial
sequenceSynthetic construct 189Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Glu Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4019040PRTArtificial sequenceSynthetic construct 190Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys His Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4019140PRTArtificial
sequenceSynthetic construct 191Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ile Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4019240PRTArtificial sequenceSynthetic construct 192Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Arg Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4019340PRTArtificial
sequenceSynthetic construct 193Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ser Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4019440PRTArtificial sequenceSynthetic construct 194Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Val Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4019540PRTArtificial
sequenceSynthetic construct 195Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Arg 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4019640PRTArtificial sequenceSynthetic construct 196Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Thr 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4019740PRTArtificial
sequenceSynthetic construct 197Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Glu Gly Phe Val Cys Lys Cys
Tyr 35 4019840PRTArtificial sequenceSynthetic construct 198Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Asn Gly Phe Val Cys Lys Cys Tyr 35 4019940PRTArtificial
sequenceSynthetic construct 199Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Ser Gly Phe Val Cys Lys Cys
Tyr 35 4020040PRTArtificial sequenceSynthetic construct 200Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Thr Gly Phe Val Cys Lys Cys Tyr 35 4020140PRTArtificial
sequenceSynthetic construct 201Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly His Phe Val Cys Lys Cys
Tyr 35 4020240PRTArtificial sequenceSynthetic construct 202Gly Phe
Gly Cys Asn
Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys
Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Trp Phe
Val Cys Lys Cys Tyr 35 4020340PRTArtificial sequenceSynthetic
construct 203Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Ala Val Cys Lys Cys Tyr 35
4020440PRTArtificial sequenceSynthetic construct 204Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
His Val Cys Lys Cys Tyr 35 4020540PRTArtificial sequenceSynthetic
construct 205Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Ile Val Cys Lys Cys Tyr 35
4020640PRTArtificial sequenceSynthetic construct 206Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Met Val Cys Lys Cys Tyr 35 4020740PRTArtificial sequenceSynthetic
construct 207Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Val Val Cys Lys Cys Tyr 35
4020840PRTArtificial sequenceSynthetic construct 208Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Trp Val Cys Lys Cys Tyr 35 4020940PRTArtificial sequenceSynthetic
construct 209Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Ile Cys Lys Cys Tyr 35
4021040PRTArtificial sequenceSynthetic construct 210Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Lys Cys Lys Cys Tyr 35 4021140PRTArtificial sequenceSynthetic
construct 211Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Gln Cys Lys Cys Tyr 35
4021240PRTArtificial sequenceSynthetic construct 212Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Arg Cys Lys Cys Tyr 35 4021340PRTArtificial sequenceSynthetic
construct 213Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys His Cys Tyr 35
4021440PRTArtificial sequenceSynthetic construct 214Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Asn Cys Tyr 35 4021540PRTArtificial sequenceSynthetic
construct 215Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Arg Cys Tyr 35
4021640PRTArtificial sequenceSynthetic construct 216Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Ile 35 4021740PRTArtificial sequenceSynthetic
construct 217Gly Phe Gly Cys Asn Gly Pro Trp Leu Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Asn Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4021840PRTArtificial sequenceSynthetic construct 218Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Leu Asp Ile Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4021940PRTArtificial sequenceSynthetic
construct 219Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Arg
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Phe Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4022040PRTArtificial sequenceSynthetic construct 220Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Arg Cys His1 5 10 15Asn His Cys
Lys Ser Ile Arg Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4022140PRTArtificial sequenceSynthetic
construct 221Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Arg Cys His1 5 10 15Asn His Cys Arg Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4022240PRTArtificial sequenceSynthetic construct 222Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Ser Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Arg Cys Tyr 35 4022340PRTArtificial sequenceSynthetic
construct 223Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Ser His Cys Lys Ser Ile Arg Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4022440PRTArtificial sequenceSynthetic construct 224Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Phe Cys Ala Arg 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4022540PRTArtificial sequenceSynthetic
construct 225Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Arg 20 25 30Gly Lys Phe Val Cys Lys Cys Tyr 35
4022640PRTArtificial sequenceSynthetic construct 226Gly Phe Gly Cys
Arg Gly Pro Trp Asp Glu Asp Asp Tyr Gln Cys His1 5 10 15Arg His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4022740PRTArtificial sequenceSynthetic
construct 227Gly Phe Gly Cys Asn Gly Pro Trp Ser Glu Asp Asp Met
Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Leu Cys Lys Cys Tyr 35
4022840PRTArtificial sequenceSynthetic construct 228Gly Phe Gly Cys
Ser Gly Pro Trp Asp Glu Asp Asp Trp Gln Cys His1 5 10 15Arg His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4022940PRTArtificial sequenceSynthetic
construct 229Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Gly
Gln Cys His1 5 10 15Arg His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Ala Gly Phe Val Cys Lys Cys Tyr 35
4023040PRTArtificial sequenceSynthetic construct 230Gly Phe Gly Cys
Ser Gly Pro Trp Ser Glu Asp Asp Leu Arg Cys His1 5 10 15Val His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ser Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4023140PRTArtificial sequenceSynthetic
construct 231Gly Phe Gly Cys Asn Gly Pro Trp Ser Glu Asp Asp Met
Leu Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4023240PRTArtificial sequenceSynthetic construct 232Gly Phe Gly Cys
Ser Gly Pro Trp Ala Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4023340PRTArtificial sequenceSynthetic
construct 233Gly Phe Gly Cys Gly Gly Pro Trp Asp Glu Asp Asp Leu
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4023440PRTArtificial sequenceSynthetic construct 234Gly Phe Gly Cys
Asn Gly Pro Trp Ala Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Arg Cys Tyr 35 4023540PRTArtificial sequenceSynthetic
construct 235Gly Phe Gly Cys Arg Gly Pro Trp Asp Glu Asp Asp Val
Gln Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4023640PRTArtificial sequenceSynthetic construct 236Gly Phe Gly Cys
Gly Gly Pro Trp Asp Glu Asp Asp Tyr Gln Cys His1 5 10 15Lys His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4023740PRTArtificial sequenceSynthetic
construct 237Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Phe
Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4023840PRTArtificial sequenceSynthetic construct 238Gly Phe Gly Cys
Ala Gly Pro Trp Ser Glu Asp Asp Leu Gln Cys His1 5 10 15Thr His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4023940PRTArtificial sequenceSynthetic
construct 239Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Thr
Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4024040PRTArtificial sequenceSynthetic construct 240Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Leu Lys Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4024140PRTArtificial sequenceSynthetic
construct 241Gly Phe Gly Cys Asn Gly Pro Trp Asn Glu Asp Asp Leu
Arg Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4024240PRTArtificial sequenceSynthetic construct 242Gly Phe Gly Cys
Asn Gly Pro Trp Ala Glu Asp Asp Met His Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Leu Cys Lys Cys Tyr 35 4024340PRTArtificial sequenceSynthetic
construct 243Gly Phe Gly Cys Ser Gly Pro Trp Ser Glu Asp Asp Val
Gln Cys His1 5 10 15Arg His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4024440PRTArtificial sequenceSynthetic construct 244Gly Phe Gly Cys
Gly Gly Pro Trp Ser Glu Asp Asp Leu Gln Cys His1 5 10 15Gln His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Thr Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4024540PRTArtificial sequenceSynthetic
construct 245Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Val
Gln Cys His1 5 10 15Thr His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4024640PRTArtificial sequenceSynthetic construct 246Gly Phe Gly Cys
Asn Gly Pro Trp Ser Glu Asp Asp Leu His Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4024740PRTArtificial sequenceSynthetic
construct 247Gly Phe Gly Cys Asn Gly Pro Trp Ser Glu Asp Asp Met
Leu Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4024840PRTArtificial sequenceSynthetic construct 248Gly Phe Gly Cys
Asn Gly Pro Trp Asn Glu Asp Asp Met His Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Arg Cys Tyr 35 4024940PRTArtificial sequenceSynthetic
construct 249Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Tyr
Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4025040PRTArtificial sequenceSynthetic construct 250Gly Phe Gly Cys
Ser Gly Pro Trp Ser Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4025140PRTArtificial sequenceSynthetic
construct 251Gly Phe Gly Cys Gly Gly Pro Trp Asp Glu Asp Asp Met
Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4025240PRTArtificial sequenceSynthetic construct 252Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Arg Lys Cys His1 5
10 15Asn His Cys Lys Ser Ile Lys Gly Tyr Arg Gly Gly Tyr Cys Ala
Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35 4025340PRTArtificial
sequenceSynthetic construct 253Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Lys His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Arg 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4025440PRTArtificial sequenceSynthetic construct 254Gly Phe
Gly Cys Gly Gly Pro Trp Asp Glu Asp Asp Trp Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4025540PRTArtificial
sequenceSynthetic construct 255Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Ala Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4025640PRTArtificial sequenceSynthetic construct 256Gly Phe
Gly Cys Lys Gly Pro Trp Asp Glu Asp Asp Leu Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4025740PRTArtificial
sequenceSynthetic construct 257Gly Phe Gly Cys Gly Gly Pro Trp Asp
Glu Asp Asp Trp Gln Cys His1 5 10 15Ser His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4025840PRTArtificial sequenceSynthetic construct 258Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Lys
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4025940PRTArtificial
sequenceSynthetic construct 259Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Asn Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4026043PRTArtificial sequenceSynthetic construct 260Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Cys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr Arg Cys Gly 35
4026140PRTArtificial sequenceSynthetic construct 261Gly Phe Gly Cys
His Gly Pro Trp Asp Glu Asp Asp Glu Gln Cys His1 5 10 15Glu His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4026240PRTArtificial sequenceSynthetic
construct 262Gly Phe Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met
Gln Cys His1 5 10 15Phe His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly
Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4026340PRTArtificial sequenceSynthetic construct 263Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Ser His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4026440PRTArtificial sequenceSynthetic
construct 264Gly Phe Gly Cys Gly Gly Pro Trp Ala Glu Asp Asp Met
Ser Cys His1 5 10 15Asn His Cys Lys Ser Ile Thr Gly Tyr Lys Gly Gly
Tyr Cys Thr Lys 20 25 30Gly Gly Phe Val Cys Lys Cys Tyr 35
4026540PRTArtificial sequenceSynthetic construct 265Gly Phe Gly Cys
Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Ala His Cys
Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly
Phe Val Cys Lys Cys Tyr 35 4026640PRTArtificialSynthetic
antimicrobial polypeptide 266Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Ser Lys Cys His1 5 10 15Asn His Cys Lys Ser Ile Lys Gly
Tyr Arg Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4026740PRTArtificial sequenceSynthetic construct 267Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Gly Asp Met Gln Cys His1 5 10 15Ile
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4026840PRTArtificial
sequenceSynthetic construct 268Gly Phe Gly Cys Ser Gly Pro Trp Asp
Glu Asp Asp Val Gln Cys His1 5 10 15Thr His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4026940PRTArtificial sequenceSynthetic construct 269Gly Phe
Gly Cys Asp Gly Pro Trp Ser Glu Asp Asp Met Arg Cys His1 5 10 15Asn
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4027040PRTArtificial
sequenceSynthetic construct 270Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Ala Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4027140PRTArtificial sequenceSynthetic construct 271Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Arg
His Cys Lys Ser Ile Lys Gly Arg Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4027240PRTArtificial
sequenceSynthetic construct 272Gly Phe Gly Cys Ser Gly Pro Trp Asp
Glu Asp Asp Val Gln Cys His1 5 10 15Ala His Cys Lys Ser Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 4027340PRTArtificial sequenceSynthetic construct 273Gly Phe
Gly Cys Asn Gly Pro Trp Asp Glu Asp Asp Met Gln Cys His1 5 10 15Thr
His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25
30Gly Gly Phe Val Cys Lys Cys Tyr 35 4027440PRTArtificial
sequenceSynthetic construct 274Gly Phe Gly Cys Asn Gly Pro Trp Asp
Glu Asp Asp Met Gln Cys His1 5 10 15Asn His Cys Lys Val Ile Lys Gly
Tyr Lys Gly Gly Tyr Cys Ala Lys 20 25 30Gly Gly Phe Val Cys Lys Cys
Tyr 35 40
* * * * *
References