U.S. patent application number 17/232470 was filed with the patent office on 2022-02-24 for n-terminal capping modules of ankyrin repeat domains.
This patent application is currently assigned to Athebio AG. The applicant listed for this patent is Athebio AG. Invention is credited to Patrik Forrer, Johannes Schilling.
Application Number | 20220056086 17/232470 |
Document ID | / |
Family ID | 1000006136222 |
Filed Date | 2022-02-24 |
United States Patent
Application |
20220056086 |
Kind Code |
A1 |
Schilling; Johannes ; et
al. |
February 24, 2022 |
N-TERMINAL CAPPING MODULES OF ANKYRIN REPEAT DOMAINS
Abstract
Described herein are proteins comprising an ankyrin repeat
domain having an N-terminal capping module with improved
properties, as well as corresponding protein libraries,
pharmaceutical compositions and nucleic acids encoding such
proteins. In other aspects, the disclosure relates to methods using
such proteins, corresponding protein libraries or pharmaceutical
compositions.
Inventors: |
Schilling; Johannes;
(Zurich-Schlieren, CH) ; Forrer; Patrik;
(Zurich-Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Athebio AG |
Zurich-Schlieren |
|
CH |
|
|
Assignee: |
Athebio AG
Zurich-Schlieren
CH
|
Family ID: |
1000006136222 |
Appl. No.: |
17/232470 |
Filed: |
April 16, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 2020 |
EP |
20191632.7 |
Claims
1. (canceled)
2. The protein according to claim 21, wherein said N-terminal
capping module comprises an amino acid sequence that has at least
90% sequence identity to the amino acid sequence of SEQ ID NO:
58.
3. The protein according to claim 21, wherein said N-terminal
capping module comprises an amino acid sequence that has at least
95% sequence identity to the amino acid sequence of SEQ ID NO:
58.
4. The protein according to claim 21, wherein the amino acid
residue at the position of said N-terminal capping module
corresponding to position 15 is L.
5. The protein according to claim 21, wherein said N-terminal
capping module comprises an amino acid sequence that has at least
95% sequence identity to the amino acid sequence of SEQ ID NO: 58;
and wherein the amino acid residue at the position of said
N-terminal capping module corresponding to position 15 is L.
6. The protein according to claim 21, wherein said N-terminal
capping module further has a dipeptide GS N-terminal to the
sequence having the at least 80% sequence identity with the amino
acid sequence of SEQ ID NO: 58.
7. (canceled)
8. The protein according to claim 21, wherein said ankyrin repeat
domain comprises one or more internal ankyrin repeats, and wherein
said one or more internal ankyrin repeats each have an amino acid
sequence having at least 80% sequence identity with the amino acid
sequence of SEQ ID NO: 81.
9. The protein according to claim 21, wherein said ankyrin repeat
domain comprises one or more internal ankyrin repeats, and wherein
said one or more internal ankyrin repeats each have an amino acid
sequence having at least 95% sequence identity with the amino acid
sequence of SEQ ID NO: 81.
10. The protein according to claim 21, wherein said ankyrin repeat
domain specifically binds to a virus.
11. The protein according to claim 21, wherein said protein
comprises one or more additional ankyrin repeat domains as further
moieties that are connected by a proline-threonine linker.
12. The protein according to claim 21, wherein said protein
comprises one or more additional ankyrin repeat domains as further
moieties that are connected by a proline-threonine linker with a
length of 2 to 24 amino acid residues.
13. The protein according to claim 21, wherein said protein
comprises at least five ankyrin repeat domains.
14. The protein according to claim 21, wherein said ankyrin repeat
domain comprises an N-terminal capping module having an amino acid
sequence that has at least 95% sequence identity with the amino
acid sequence of SEQ ID NO: 58, wherein the amino acid residue at
the position of said N-terminal capping module corresponding to
position 15 is L; wherein said N-terminal capping module further
has a dipeptide GS N-terminal to the sequence having the at least
95% sequence identity with the amino acid sequence of SEQ ID NO:
58; and wherein said ankyrin repeat domain comprises one or more
internal ankyrin repeats that each have an amino acid sequence
having at least 95% sequence identity with the amino acid sequence
of SEQ ID NO: 81.
15. The protein according to claim 14, wherein said protein
comprises one or more additional ankyrin repeat domains as further
moieties that are connected by a proline-threonine linker with a
length of 2 to 24 amino acid residues; and wherein said protein
comprises at least five ankyrin repeat domains.
16. The protein according to claim 14, wherein said ankyrin repeat
domain specifically binds to a virus.
17. (canceled)
18. (canceled)
19. (canceled)
20. A method of preparing a protein comprising an ankyrin repeat
domain with an improved thermostability, the method comprising the
following steps (A) or (B): (A) (i) providing a library with more
than one protein, each protein comprising an ankyrin repeat domain
that comprises an N-terminal capping module, one or more internal
ankyrin repeat(s) and a C-terminal capping module, and (ii)
selecting a protein having binding specificity to a target from the
library of proteins of step (i), wherein the N-terminal capping
module of the ankyrin repeat domain of said selected protein has an
amino acid residue selected from the group consisting of I, T, A,
V, L and M at the position corresponding to position 15 and
comprises an amino acid sequence that has at least 80% sequence
identity with the amino acid sequence of SEQ ID NO: 58, and wherein
the thermostability of the ankyrin repeat domain of said selected
protein is improved in comparison to a reference ankyrin repeat
domain having the same amino acid sequence except for the amino
acid residue at the position of the N-terminal capping module
corresponding to position 15, which is D in the reference ankyrin
repeat domain; or alternatively, (B) (i) selecting a protein
comprising an ankyrin repeat domain that comprises an N-terminal
capping module, one or more internal ankyrin repeat(s) and a
C-terminal capping module, and (ii) replacing the N-terminal
capping module of said ankyrin repeat domain by an N-terminal
capping module that has an amino acid residue selected from the
group consisting of I, T, A, V, L and M at the position
corresponding to position 15 and comprises an amino acid sequence
that has at least 80% sequence identity with the amino acid
sequence of SEQ ID NO: 58, wherein the thermostability of said
ankyrin repeat domain with the replaced N-terminal capping module
is improved in comparison to a reference ankyrin repeat domain
having the same amino acid sequence except for the amino acid
residue at the position of the N-terminal capping module
corresponding to position 15, which is D in the reference ankyrin
repeat domain.
21. A protein comprising an ankyrin repeat domain obtained by the
method according to claim 20.
22. The method according to claim 20, wherein the N-terminal
capping module of the ankyrin repeat domain of said selected
protein according to the method of claim 20 (A) or the N-terminal
capping module of said ankyrin repeat domain with the replaced
N-terminal capping module according to the method of claim 20 (B)
comprises an amino acid sequence has at least 90% sequence identity
with the amino acid sequence of SEQ ID NO: 58.
23. The method according to claim 20, wherein the N-terminal
capping module of the ankyrin repeat domain of said selected
protein according to the method of claim 20 (A) or the N-terminal
capping module of said ankyrin repeat domain with the replaced
N-terminal capping module according to the method of claim 20 (B)
comprises an amino acid sequence has at least 95% sequence identity
with the amino acid sequence of SEQ ID NO: 58.
24. The method according to claim 23, wherein the N-terminal
capping module of the ankyrin repeat domain of said selected
protein according to the method of claim 20 (A) or the N-terminal
capping module of said ankyrin repeat domain with the replaced
N-terminal capping module according to the method of claim 20 (B)
has L at the position corresponding to position 15 and a dipeptide
GS to N-terminal to the sequence having the at least 95% sequence
identity with the amino acid sequence of SEQ ID NO: 58; and wherein
said ankyrin repeat domain of said selected protein according to
the method of claim 20 (A) or said ankyrin repeat domain with the
replaced N-terminal capping module according to the method of claim
20 (B) comprises one or more internal ankyrin repeats that each
have an amino acid sequence having at least 95% sequence identity
with the amino acid sequence of SEQ ID NO: 81.
25. The method according to claim 20, the method comprising the
step (A).
26. The method according to claim 20, the method comprising the
step (B).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(a) of EP Application No. EP20191632.7 filed Aug. 18, 2020, the
contents of which are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The sequence listing of the present application has been
submitted electronically via EFS-Web as an ASCII formatted sequence
listing with a file name
"2021-04-16-Sequence-Listing-Athebio-AG-190010USPT.txt", creation
date of Apr. 16, 2021, and a size of 70,986 bytes. The sequence
listing submitted via EFS-Web is part of the specification and is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to proteins comprising an
ankyrin repeat domain with an N-terminal capping module conferring
improved properties to the ankyrin repeat domain, as well as
related products and the use of such proteins in methods of
treatment.
BACKGROUND OF THE INVENTION
[0004] Different classes of specific binding proteins have evolved
in nature, the most widely known class being immunoglobulins of
vertebrates. Another class of specific binding proteins are repeat
proteins. Similar to the role that immunoglobulins play in
vertebrates, repeat proteins were found to be involved in the
adaptive immune system of jawless fish. However, repeat proteins
play a much wider role beyond this function and mediate
protein-protein interactions across all phyla to fulfill diverse
biological functions. In fact, they constitute the largest group of
natural proteins mediating specific binding (e.g. reviewed in
Forrer, P., et al., FEBS letters 539, 2-6, 2003). Repeat proteins
bind their targets via the repeat domain, which is made up of a
variable number of repeats that stack on each other through their
conserved interfaces to create the compactly folded repeat domain.
Specific target binding is then achieved through variable residues
on the surface of the repeat domain (Forrer 2003, loc. cit. and WO
2002/020565).
[0005] Ankyrin repeat proteins are a well-studied class of repeat
proteins. The ankyrin repeat usually comprises 33 amino acid
residues forming two antiparallel alpha-helices and a beta-turn.
The folded ankyrin repeat domain comprising the stacked ankyrin
repeats has a right-handed solenoid structure with a compact
hydrophobic core and a large binding surface, which allows it to
adapt to its respective binding partners (e.g. Binz, H. K., et al.,
Nat. Biotechnol. 22, 575-582, 2004).
[0006] Pluckthun and coworkers derived a consensus sequence motif
from naturally occurring ankyrin repeats (e.g., Binz, H. K., et
al., J. Mol. Biol., 332, 489-503, 2003 and WO 2002/020565). The
derived ankyrin repeat consensus motif is 33-amino acid residues
long and comprises fixed and variable positions. The fixed
positions correspond mainly to framework residues that are
primarily responsible for the structural integrity of the ankyrin
repeats, in particular, for the interrepeat stacking interactions.
The variable positions correspond to surface-exposed residues that
do not strongly contribute to the structural integrity of the
ankyrin repeats; but, are potentially involved in target binding
(though surface-exposed framework residues may be involved in
target binding too).
[0007] Libraries of proteins were then created having an ankyrin
repeat domain with internal ankyrin repeats that were based on such
ankyrin repeat consensus motif (Binz, 2004, loc. cit.). Certain
variable positions of the consensus motif were randomized in each
internal ankyrin repeat to allow for the binding to different
targets, thereby creating the diversity of the library. In order to
avoid aggregation of ankyrin repeat domains consisting only of
internal ankyrin repeats, the internal ankyrin repeats were flanked
by an N-terminal capping module and a C-terminal capping module to
shield the hydrophobic core of the domain from the solvent (Forrer,
2003, loc. cit. and Binz, 2003, loc. cit.). These capping modules
were based on the capping repeats of the human
guanine-adenine-binding protein (GA-binding protein). Libraries
composed of proteins with ankyrin repeat domains having one, two or
three internal ankyrin repeats are referred to as an N1C, N2C and
N3C library, respectively, wherein the "N" refers to the N-terminal
capping module, "C" refers to the C-terminal capping module and the
number to the number of internal ankyrin repeats (Binz, 2003, loc.
cit).
[0008] Using such a synthetic library of designed ankyrin repeat
proteins (DARPins), DARPins against specific targets can be
selected with common selection methods, including phage display,
ribosome display and yeast display, and were shown to have very
favorable properties. While displaying binding specificities and
affinities that are comparable to immunoglobulins, DARPins are much
more robust and can be easily engineered into multispecific binding
proteins that are easily expressed and purified. DARPins are well
studied (e.g. Pluckthun, A., Annu. Rev. Pharmacol. Toxicol. 55,
489-511, 2015).
[0009] Following the design of the original DARPin library by
Pluckthun and coworkers (Binz, 2003, loc. cit. and WO 2002/020565),
it was shown that a mutation in the N-terminal capping module can
increase the thermostability of an ankyrin repeat domain (WO
2012/069655).
[0010] There remains a need to further improve the properties of
proteins comprising an ankyrin repeat domain, such as the
thermostability of the ankyrin repeat domain.
SUMMARY OF THE INVENTION
[0011] The present invention is based on the discovery that
specific mutations in the N-terminal capping module can
significantly improve the properties of an ankyrin repeat domain.
In particular, it has been found that the amino acid residue
present at the position in the N-terminal capping module, which
corresponds to position 15 of SEQ ID NO: 3, is of key importance
for the thermostability of an ankyrin repeat domain. It has also
been found that only a few amino acid residues in this position
have a striking effect on thermostability. Furthermore, the effects
of these mutations were transferable to ankyrin repeat domains with
diverging sequences, demonstrating the general importance of this
position in the N-terminal capping module for the thermostability
of ankyrin repeat domains.
[0012] Accordingly, the present invention provides a protein
comprising an ankyrin repeat domain, wherein the ankyrin repeat
domain comprises an N-terminal capping module with a mutation in
the position, which corresponds to position 15 of SEQ ID NO: 3.
[0013] In a further aspect, the present invention provides a
protein library comprising such proteins.
[0014] In a further aspect, the present invention provides a method
of selection using such protein libraries.
[0015] The present invention also provides pharmaceutical
compositions comprising the proteins of the invention, nucleic
acids encoding them or vectors or cells comprising said nucleic
acids.
[0016] In a further aspect, the present invention provides a method
of preparing a protein of the invention comprising culturing a cell
having a nucleic acid encoding the protein of the invention under
conditions allowing expression thereof and then purifying the
expressed protein.
[0017] In a further aspect, the present invention relates to the
proteins of the invention for use in a method of treatment.
[0018] Related compositions and methods are also provided, as will
be apparent from the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1: Thermal stability of the designed ankyrin repeat
proteins P #95 and P #101.
[0020] Traces from thermal denaturation of proteins P #95 and P
#101 are shown. The thermal denaturation is followed by the CD
signal at 222 nm in PBS at pH 7.4. The Tm values for P #95 and P
#101 were estimated to be 74.5.degree. C. and 85.1.degree. C.,
respectively.
[0021] FF, fraction folded in %; T, temperature in .degree. C.; P
#95 and P #101 consist of the amino acid sequences of SEQ ID NO: 95
and SEQ ID NO: 101, respectively. P #101, but not P #95, comprises
an improved N-terminal capping module of the invention.
[0022] FIG. 2: Thermal stability of the designed ankyrin repeat
proteins P #93 and P #94.
[0023] Traces from thermal denaturation of P #93 and P #94 are
shown. The thermal denaturation is followed by the CD signal at 222
nm in PBS at pH 7.4. The Tm values for P #93 and P #94 were
estimated to be 62.1.degree. C. and 75.2.degree. C.,
respectively.
[0024] FF, fraction folded in %; T, temperature in .degree. C.; P
#93 and P #94 consist of the amino acid sequences of SEQ ID NO:93
and SEQ ID NO:94, respectively. P #94, but not P #93, comprises an
improved N-terminal capping module of the invention.
[0025] FIG. 3: Thermal stability of the designed ankyrin repeat
proteins P #108 and P #109.
[0026] Traces from thermal denaturation of proteins P #108 and P
#109 are shown. The thermal denaturation is followed by the CD
signal at 222 nm in PBS at pH 7.4. The Tm values for P #108 and P
#109 were estimated to be 68.6.degree. C. and 82.8.degree. C.,
respectively.
[0027] FF, fraction folded in %; T, temperature in .degree. C.; P
#108 and P #109 consist of the amino acid sequences of SEQ ID NO:
108 and SEQ ID NO: 109, respectively. P #109, but not P #108,
comprises an improved N-terminal capping module of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0028] "A", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
a protein comprising an ankyrin repeat domain refers to one or more
such proteins.
[0029] An "ankyrin repeat" refers to a short sequence of amino acid
residues forming a structural motif (e.g., SMART accession number:
SM00248). Ankyrin repeats occur in consecutive copies, are involved
in protein-protein interactions and the core of the ankyrin repeat
forms a helix-loop-helix structure.
[0030] The term "ankyrin repeat domain" refers to a protein domain
comprising an N-terminal capping module, a C-terminal capping
module and one or more ankyrin repeats in between (also referred to
as "internal ankyrin repeats"). An ankyrin repeat domain has a
hydrophobic core, which is shielded on its N- and C-terminal end by
N- and C-terminal capping modules, respectively, from the solvent.
The folded ankyrin repeat domain has a right-handed solenoid
structure with a large binding surface that is adaptable to
specifically bind targets. The ankyrin repeat domain is generally
very robust and can sustain a significant number of mutations,
including substitutions, additions and deletions, without
destroying its overall structure. The residues that contribute to
the structural integrity of the ankyrin repeat domain, including
the interrepeat interactions, are referred to as "framework
residues", whereas the residues that contribute to target binding,
either through direct interaction with the target or by influencing
residues that directly interact with the target, e.g., by
stabilizing them, are referred to as "target interaction residues".
A single amino acid residue can be both--a framework and a target
interaction residue--at the same time and framework residues and
target interaction residues may be found not only in the internal
ankyrin repeats, but also the N-terminal capping module and/or the
C-terminal capping module.
[0031] The internal ankyrin repeats contribute to the structural
stability of the ankyrin repeat domain due to their stacking
interaction with the neighboring repeats. An internal ankyrin
repeat usually consists of 33 amino acid residues.
[0032] The capping modules have a hydrophobic inside surface that
is suitable for interacting with the adjacent internal ankyrin
repeat and a hydrophilic outside surface to shield the hydrophobic
core from the solvent. In some embodiments, the N-terminal capping
module and/or the C-terminal capping module are a N-terminal
capping repeat and/or C-terminal capping repeat, respectively,
which have a similar or the same fold as the adjacent internal
ankyrin repeat(s) and/or sequence similarities to said adjacent
internal ankyrin repeat(s).
[0033] The terms "binding", "specific binding" or the like when
used in reference to a target mean a binding interaction that is
measurably different from a non-specific interaction, e.g., the
interaction with a control molecule that is unrelated to the
specific target. Control molecules that are commonly used to
measure such non-specific interaction include bovine serum albumin,
bovine casein and Escherichia coli maltose binding protein (unless,
of course, the respective proteins were the target for the specific
binding). In certain instances, the terms "binding", "specific
binding" or the like mean that only the target is bound and
substantially no other molecule. Specific binding can be
determined, for instance, by measuring the dissociation constant
(Kd) for the target and/or by comparing the binding to the target
with the binding to a control molecule. The Kd can be measured by
various conventional techniques, such as isothermal titration
calorimetry, radioligand binding assay, fluorescence energy
resonance transfer, and surface plasmon resonance. The binding
specificity is generally measured in standardized solutions, such
as PBS. For instance, the Kd for the target in PBS is at least 10,
at least 10.sup.2, at least 10.sup.3 or at least 10.sup.4 times
lower than the corresponding Kd for a control molecule that is
unrelated to the specific target.
[0034] The term "designed ankyrin repeat protein" or "DARPin"
refers to a non-natural protein comprising an ankyrin repeat
domain. In some embodiments, such a DARPin has a repeat sequence
motif that was derived from natural ankyrin repeats, e.g. by
consensus design (see, e.g., Forrer et al., 2004 Chem Bio Chem, 5,
2, 183-189 and Binz 2003, loc. cit).
[0035] The term "fraction of refolded ankyrin repeat domains after
thermal denaturation" refers to the fraction of ankyrin repeat
domains that refold into their native state after thermal
denaturation.
[0036] The term "library" as used in reference to a protein or
nucleic acid library refers to a collection of proteins and nucleic
acids, respectively.
[0037] The term "melting temperature" or "Tm" refers to the
temperature at which 50% of the protein is unfolded in a certain
buffer, e.g. PBS.
[0038] The term "modification", as used in reference to a specific
amino acid sequence (e.g. the amino acid sequence of an internal
ankyrin repeat or capping module), refers to one or more
modification(s) of said amino acid sequence selected from the group
consisting of deletions, insertions and/or substitutions. In some
embodiments, the number of deletions and insertions is limited, for
instance, to a combined number of deletions and insertions of not
more than three, not more than two or not more than one of the
total number of modification(s). Accordingly, if there is a total
number of not more than 9 modifications, the number of deletions
and insertions of those not more than 9 modifications may be
limited to a combined number of insertions and deletions of not
more than two. In some embodiments, the modification(s) are
substitution(s) only. A substitution can be a substitution of an
amino acid residue with, e.g., any of the naturally occurring amino
acid residues. In some embodiments, the substitution of an amino
acid residue is with an amino acid residue selected from the group
consisting of A, D, E, F, H, I, K, L, M, N, Q, R, S, V, W and Y. In
some embodiments, the substitution of an amino acid residue is with
an amino acid residue selected from the group consisting of A, D,
E, H, I, K, L, Q, R, S, V and Y. The following amino acid residues
may, for instance, be particularly suitable for the respective
position of the N-terminal capping module:
TABLE-US-00001 TABLE 1 Position Amino acid residue 1 A, E, N, Q, G,
S, T, K, D, R, H, C 2 E, L, Q, M, K, R, C 3 G, D, S, A, C 4 A, R,
T, S, N, Q, K, D, E, H, C 5 A, R, T, S, N, Q, K, D, E, H, C 6 A, L,
N, S, D, C 7 L, I, V, A, N, T, S, D, C 8 E, D, Q, A, N, S, T, K, R,
H, C 9 A 10 V, S, A, C 11 L, S, Q, K, R, A, H, D, E, C, T, N, F, W,
Y 12 K, R, A, T, S, N, Q, D, E, H, C 13 G, C 14 N, S, T, A, D, E,
K, Q, R, H, C 15 M, I, T, A, L, V, S, N, D, Q, K, R, E, C 16 D, A,
N, Q, S, T, K, E, R, H, C 17 D, A, N, Q, S, T, K, E, R, H, C 18 T,
A, S, I, L, V, C 19 R, E, D, K, A, N, Q, S, T, H, C 20 N, K, R, T,
S, E, Q, A, D, H, C, I, V 21 N, S, L, A, C 22 I, A, V, M, T, L, S,
N, C 23 R, S, Q, K, N, A, E, D, H, C 24 A, H, K, R, L, I, V, C, G
25 G 26 A 27 N, D, C 28 T, V, S, P, A, C 29 D, N, C 30 A, C
[0039] An amino acid substitution may be a conservative or
non-conservative substitution. In some embodiments, substitutions
only relate to conservative amino acid substitutions. A
conservative amino acid substitution typically involves exchanging
an amino acid residue by a different one having similar biophysical
properties. For instance, the amino acid residues with a positively
charged sidechain K, R and H, the amido acids with negatively
charged sidechain E and D, the amino acid residue with a polar side
chain T and S, the amino acid residues with an aromatic sidechain
F, W or Y or the amino acid residues with a non-polar sidechain A,
V, L, I and M may be substituted with one another.
[0040] The term "PBS" refers to phosphate-buffered saline
containing 137 mM NaCl, 10 mM phosphate and 2.7 mM KCl and having a
pH of 7.4.
[0041] The term "percent (%) sequence identity" with respect to a
specific amino acid sequence (e.g. the amino acid sequence of a
N-terminal capping module of the invention) is defined as the
percentage of amino acid residues in a candidate sequence that is
identical with the amino acid residues in the specific amino acid
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2 or ALIGN. Those
skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being
compared. Further examples of how to determine the percentage of
sequence identity can be found in WO 2009/058564 A2, page 93, line
14 to page 102, line 5.
[0042] The term "pharmaceutically acceptable carrier" refers to
buffers, carriers, and other excipients suitable for use in contact
with tissues of humans and/or animals without excessive toxicity,
allergic response, irritation, or other problem or complication,
commensurate with a reasonable benefit/risk ratio. The carrier(s)
should be "acceptable" in the sense of being compatible with the
other ingredients of the formulations and not deleterious to the
recipient. Pharmaceutically acceptable carriers include buffers,
solvents, dispersion media, coatings, isotonic and absorption
delaying agents, and the like, that are compatible with
pharmaceutical administration.
[0043] The term "pharmaceutical composition" refers to a
composition comprising at least one active agent and at least one
pharmaceutically acceptable carrier. A pharmaceutical composition
is generally formulated and administered to exert a
pharmaceutically useful effect while minimizing undesirable side
effects.
[0044] If the term "position" is used without further reference to
a particular amino acid sequence, then it refers to the
corresponding amino acid position of SEQ ID NO: 3. Furthermore,
"corresponding" in this context means that the amino acid residue
aligns with the indicated position of a specific sequence in a
sequence alignment. Alignment for purposes of determining which
amino acid residue corresponds to which position of a specific
sequence can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2 or ALIGN. Those skilled in the art
can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full length of the sequences being compared. Further examples
of how to align two sequences can be found in WO 2009/058564 A2,
page 94, line 7 to page 96, line 28.
[0045] The term "recombinant", as used in reference to a protein,
refers to a protein produced from a recombinant nucleic acid. A
"recombinant nucleic acid" refers to nucleic acid molecules formed
by laboratory methods of genetic recombination or gene
synthesis.
[0046] The term "substantially identical", as used in reference to
a specific amino acid sequence (e.g. the N-terminal capping module
of the invention or an internal ankyrin repeat), refers to amino
acid sequences having (1) at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, or 100% sequence identity
to the specific amino acid sequence or (2) up to 9, up to 8, up to
7, up to 6, up to 5, up to 4, up to 3, up to 2, up to 1 or no
modifications, as compared to the specific amino acid sequence.
[0047] The term "target", as used, for instance, in conjunction
with the specific binding property of an ankyrin repeat domain
comprised in a protein, refers to any substance or structure. It
may refer to a single molecule, such as a protein, small-molecule
or sugar, as well as complexed molecules, such as interacting
proteins or proteins binding to non-proteinaceous compounds. It may
also refer to more macromolecular structures, such as cells,
tissues, viruses or bacteria.
[0048] The terms "treating" or "treatment" of a disease, condition
or symptom refers to obtaining therapeutic and/or prophylactic
benefit, including alleviating, ablating, ameliorating, or
preventing a disease, condition or symptoms, preventing additional
symptoms, ameliorating or preventing the underlying metabolic
causes of symptoms, inhibiting the disease or condition, e.g.,
arresting or slowing down the development of the disease or
condition, relieving the disease or condition, causing regression
of the disease or condition, relieving a condition caused by the
disease or condition, or stopping the symptoms of the disease or
condition.
[0049] Naturally occurring ankyrin repeat domains normally have
capping modules to shield their hydrophobic cores from the solvent.
In line with this, earlier developed recombinant proteins having
ankyrin repeat domains likewise comprised capping modules with such
a shielding function at the N- and C-terminal ends of the ankyrin
repeat domain (e.g. Binz, 2003, loc. cit. and Binz, 2004, loc.
cit.). The capping modules that were first used were derived from
the murine GABP.beta.1, which is a naturally occurring ankyrin
repeat protein (PDB: 1AWC_B). Binz et al. 2003 (loc. cit.) already
introduced some modifications to the naturally occurring capping
modules in order to adapt the capping modules for binding to the
internal ankyrin repeats and for cloning purposes. The N-terminal
capping module of Binz et al. 2003 (loc. cit.) is reflected by SEQ
ID NO: 3 and the C-terminal capping module of Binz et al. 2003
(loc. cit.) is reflected by SEQ ID NO: 83.
[0050] WO 2012/069655 relates to further modified N-terminal and
C-terminal capping modules of the ankyrin repeat domain. The
N-terminal capping modules disclosed in WO 2012/069655 include the
two N-terminal capping modules reflected by SEQ ID NO: 1 and SEQ ID
NO: 2 (corresponding to SEQ ID NO: 15 and 14 of WO 2012/069655,
respectively, without the two optionally missing N-terminal amino
acid residues). Another exemplary N-terminal capping module
disclosed in the prior art includes the one reflected by SEQ ID NO:
4 corresponding to an amino acid sequence comprised in the
N-terminal capping module of SEQ ID NO: 56 of WO 2016/156596
A1.
[0051] Using in silico structure analysis, the present inventors
determined those amino acid residues that would appear to be most
suitable for each position of the N-terminal capping module of the
ankyrin repeat domain. In particular, the various amino acid
residues shown in Table 1 were considered to be particularly
suitable based on the in silico analysis.
[0052] Based on the structural analysis, amino acid residues in
various positions were tested by in vitro experimentation. Among
the many tested mutations of the N-terminal capping module, there
was one position which was surprisingly found to be particularly
important for the thermostability of the ankyrin repeat domain--the
position corresponding to position 15 of SEQ ID NO: 3 (also simply
referred to as position 15 herein without referencing SEQ ID NO:
3).
[0053] Accordingly, the protein of the invention comprises an
ankyrin repeat domain that has an N-terminal capping module with a
mutation in position 15.
[0054] In some embodiments, the ankyrin repeat domain of the
protein of the invention has improved properties, which may include
improved thermostability, improved storage stability, improved
thermodynamic stability (defined as the difference in free energy
between the folded and unfolded states), improved folding and/or
refolding properties (such as a higher fraction of refolded ankyrin
repeat domains after thermal denaturation), reduced aggregation
propensity and lower in vivo immunogenicity risk.
[0055] In some embodiments, the N-terminal capping module has an
amino acid residue at position 15 selected from the group
consisting of I, T, A, V, L, M, S, Q and R. In some embodiments,
the N-terminal capping module has an amino acid residue at position
15 selected from the group consisting of I, T, A, V, L, M and S. In
some embodiments, the N-terminal capping module has an amino acid
residue at position 15 selected from the group consisting of I, T,
A, V, L and M. In some embodiments, the N-terminal capping module
has I at position 15. In some embodiments, the N-terminal capping
module has T at position 15. In some embodiments, the N-terminal
capping module has A at position 15. In some embodiments, the
N-terminal capping module has V at position 15. In some
embodiments, the N-terminal capping module has L at position 15. In
some embodiments, the N-terminal capping module has M at position
15.
[0056] In some embodiments, the N-terminal capping module has,
apart from the mutation in position 15, an amino acid residue of
Table 1 in one or more of the other positions.
[0057] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 3 selected from the group
consisting of G, A, D and S. In some embodiments, the N-terminal
capping module has an amino acid residue at position 3 selected
from A and S. In some embodiments, the N-terminal capping module
has A at position 3. In some embodiments, the N-terminal capping
module has S at position 3.
[0058] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 8 selected from the group
consisting of E, D, Q, N, S, T, K and R. In some embodiments, the
N-terminal capping module has Q at position 8.
[0059] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 11 selected from the group
consisting of L, S, Q, K, R, A, H, D and E. In some embodiments,
the N-terminal capping module has an amino acid residue at position
11 selected from K, E, Q, A and L. In some embodiments, the
N-terminal capping module has A at position 11.
[0060] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 16 selected from the group
consisting of D, A, N, Q, G, S, T, K, E and R. In some embodiments,
the N-terminal capping module has E at position 16.
[0061] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 17 selected from the group
consisting of D, A, N, Q, G, S, T, K, E and R. In some embodiments,
the N-terminal capping module has A at position 17.
[0062] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 19 selected from the group
consisting of R, E, D, K, A, N, Q, S, T and H. In some embodiments,
the N-terminal capping module has E at position 19.
[0063] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 20 selected from the group
consisting of I, V, N, K, R, T, S, E, Q, A, D and H. In some
embodiments, the N-terminal capping module has Q at position
20.
[0064] In some embodiments, the N-terminal capping module further
has an amino acid residue at position 22 selected from the group
consisting of L, V, I and A. In some embodiments, the N-terminal
capping module further has an amino acid residue at position 22
selected from the group consisting of L, V and I. In some
embodiments, the N-terminal capping module has L at position 22. In
some embodiments, the N-terminal capping module has V at position
22. In some embodiments, the N-terminal capping module has I at
position 22. In some embodiments, the N-terminal capping module has
A at position 22.
[0065] In some embodiments, the N-terminal capping module has L at
position 15 and I at position 22. In some embodiments, the
N-terminal capping module has M at position 15 and I at position
22. In some embodiments, the N-terminal capping module has T at
position 15 and I at position 22. In some embodiments, the
N-terminal capping module has I at position 15 and I at position
22. In some embodiments, the N-terminal capping module has A at
position 15 and I at position 22. In some embodiments, the
N-terminal capping module has V at position 15 and I at position
22.
[0066] In some embodiments, the N-terminal capping module has L at
position 15 and L at position 22. In some embodiments, the
N-terminal capping module has M at position 15 and L at position
22. In some embodiments, the N-terminal capping module has T at
position 15 and L at position 22. In some embodiments, the
N-terminal capping module has I at position 15 and L at position
22. In some embodiments, the N-terminal capping module has A at
position 15 and L at position 22. In some embodiments, the
N-terminal capping module has V at position 15 and L at position
22.
[0067] In some embodiments, the N-terminal capping module has L at
position 15 and V at position 22. In some embodiments, the
N-terminal capping module has M at position 15 and V at position
22. In some embodiments, the N-terminal capping module has T at
position 15 and V at position 22. In some embodiments, the
N-terminal capping module has I at position 15 and V at position
22. In some embodiments, the N-terminal capping module has A at
position 15 and V at position 22. In some embodiments, the
N-terminal capping module has V at position 15 and V at position
22.
[0068] In some embodiments, the ankyrin repeat domain of the
protein of the invention has an improved thermostability, such as a
higher melting temperature and/or a higher fraction of refolded
ankyrin repeat domains after thermal denaturation, as compared to a
reference ankyrin repeat domain having the same amino acid sequence
except for the mutation in position 15 of the N-terminal capping
module.
[0069] Methods for measuring the thermostability of a protein or a
protein domain are well-known to the person skilled in the art. For
instance, the thermostability can be measured by a thermal shift
assay, differential scanning calorimetry and circular dichroism
(CD). Another possible approach is to use differential scanning
fluorimetry (e.g. Nielsen et al., 2007, Nat Protoc. 2, 9:2212-21).
In this method, unfolding of the protein is measured with a
fluorescent dye that binds to hydrophobic parts of the protein. As
the protein unfolds, more hydrophobic parts become exposed causing
an increase in fluorescence and vice versa.
[0070] This method therefore allows to conveniently monitor the
refolding properties of a protein and to determine its melting
temperature, which corresponds to the midpoint of the fluorescence
transition curve. The refolding properties and melting temperature
of a protein can also be measured by CD spectroscopy, whereby the
thermal melting curve of the protein is determined by measuring the
CD signal at 222 nm. For purposes of measuring the thermostability,
the protein may be dissolved in PBS. For example, the
thermostability of a helical protein can be determined by measuring
the CD signal of the protein at 222 nm while slowly heating the
protein at a concentration of 0.01 mM in PBS pH 7.4 from 20.degree.
C. to 95.degree. C. using a temperature ramp of 1.degree. C. per
min.
[0071] In some embodiments, the increase in melting temperature of
the ankyrin repeat domain of the invention is at least 1.degree.
C., at least 2.degree. C., at least 3.degree. C., at least
4.degree. C. or at least 5.degree. C., as compared to the reference
ankyrin repeat domain.
[0072] In some embodiments, the fraction of the refolded ankyrin
repeat domains after thermal denaturation is at least 1%, at least
5%, at least 10% or at least 20% higher, as compared to the
reference ankyrin repeat domain.
[0073] In some embodiments, the ankyrin repeat domain has a higher
melting temperature and/or higher fraction of refolded ankyrin
repeat domains after thermal denaturation. In some embodiments, the
ankyrin repeat domain has a higher melting temperature and/or
higher fraction of refolded ankyrin repeat domains after thermal
denaturation than a reference ankyrin repeat domain with the same
amino acid sequence except for position 15 of the N-terminal
capping module that is a different amino acid residue in the
reference ankyrin repeat domain compared to the ankyrin repeat
domain of the protein of the invention. In some embodiments, the
amino acid residue in position 15 of the reference ankyrin repeat
domain is selected from the group consisting of E, D, G, H, K and
N. In some embodiments, the amino acid residue in position 15 of
the reference ankyrin repeat domain is E. In some embodiments, the
amino acid residue in position 15 of the reference ankyrin repeat
domain is D.
[0074] Unless specified, the sequence of the ankyrin repeat domain
is not particularly limited. In particular, the ankyrin repeat
domain allows for a large sequence variation while preserving the
overall structure and function of the domain.
[0075] In some embodiments, the N-terminal capping module comprises
an amino acid sequence that is substantially identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1
to 76 and 110. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 17 to 26. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 27 to 36. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 37 to 46. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 47 to 56. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 57 to 66 and 110. In some embodiments, the N-terminal capping
module comprises an amino acid sequence that is substantially
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 67 to 76.
[0076] In some embodiments, the N-terminal capping module comprises
an amino acid sequence that has at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or 100%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1 to 76 and 110. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16.
In some embodiments, the N-terminal capping module comprises an
amino acid sequence that has at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or 100% sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 17 to 26. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 27 to
36. In some embodiments, the N-terminal capping module comprises an
amino acid sequence that has at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or 100% sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 37 to 46. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 47 to
56. In some embodiments, the N-terminal capping module comprises an
amino acid sequence that has at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or 100% sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 57 to 66 and 110. In some embodiments,
the N-terminal capping module comprises an amino acid sequence that
has at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 67 to
76.
[0077] In some embodiments, the N-terminal capping module comprises
an amino acid sequence that is identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 76 and 110.
In some embodiments, the N-terminal capping module comprises an
amino acid sequence that is identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16. In some
embodiments, the N-terminal capping module comprises an amino acid
sequence that is identical to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 17 to 26. In some embodiments,
the N-terminal capping module comprises an amino acid sequence that
is identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 27 to 36. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that is
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 37 to 46. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that is
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 47 to 56. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that is
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 57 to 66 and 110. In some embodiments,
the N-terminal capping module comprises an amino acid sequence that
is identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 67 to 76. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that is
identical to SEQ ID NO: 110.
[0078] In some embodiments, the N-terminal capping module comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 or from a variant of any one of SEQ ID NOs: 7 to 16
with up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3,
up to 2 or up to 1 modifications outside position 15, and wherein X
at position 15 of SEQ ID NOs: 7 to 16 or said variants thereof is
an amino acid residue as defined above for position 15. In some
embodiments, the variants of SEQ ID NOs: 7 to 16 have up to 9
modifications. In some embodiments, the variants of SEQ ID NOs: 7
to 16 have up to 8 modifications. In some embodiments, the variants
of SEQ ID NOs: 7 to 16 have up to 7 modifications. In some
embodiments, the variants of SEQ ID NOs: 7 to 16 have up to 6
modifications. In some embodiments, the variants of SEQ ID NOs: 7
to 16 have up to 5 modifications. In some embodiments, the variants
of SEQ ID NOs: 7 to 16 have up to 4 modifications. In some
embodiments, the variants of SEQ ID NOs: 7 to 16 have up to 3
modifications. In some embodiments, the variants of SEQ ID NOs: 7
to 16 have up to 2 modifications. In some embodiments, the variants
of SEQ ID NOs: 7 to 16 have up to 1 modification. In some
embodiments, there are no variants of SEQ ID NOs: 7 to 16. In some
embodiments, the modifications of SEQ ID NOs: 7 to 16 do not
include more than a combined number of deletions and insertions of
3. In some embodiments, the modifications of SEQ ID NOs: 7 to 16 do
not include more than a combined number of deletions and insertions
of 2. In some embodiments, the modifications of SEQ ID NOs: 7 to 16
do not include more than a combined number of deletions and
insertions of 1. In some embodiments, the modifications of SEQ ID
NOs: 7 to 16 are only substitutions. In some embodiments, the
variants of SEQ ID NOs: 7 to 16 comprise one or more substitutions
with an amino acid residue of Table 1.
[0079] In some embodiments, the N-terminal capping module comprises
an amino acid sequence with amino acid residues as indicated for
the positions 1 to 30 in Table 1. For instance, the amino acid
residue at position 1 is selected from the group consisting of A,
E, N, Q, G, S, T, K, D, R and H, and so on. In some embodiments,
the N-terminal capping module comprises an amino acid sequence with
amino acid residues as indicated for the positions 1 to 30 in Table
1 except for position 15 that is selected from the group consisting
of I, T, A, V, L and M. In some embodiments, the N-terminal capping
module comprises an amino acid sequence with amino acid residues as
indicated for the positions 1 to 30 in Table 1 except for position
15 that is selected from the group consisting of I, T, A, V, L and
M or a variant thereof with up to 9, up to 8, up to 7, up to 6, up
to 5, up to 4, up to 3, up to 2 or up to 1 modifications outside
position 15.
[0080] The N-terminal capping module may further comprise a
sequence N-terminal to the amino acid sequences defined in SEQ ID
NOs: 1 to 76 and 110 (or the sequence variants thereof defined
herein). For instance, such sequence could be a dipeptide
comprising amino acid residues selected from the group consisting
of D, A, E, N, Q, S, T, K, R and H, such as the dipeptide GS, DA,
EA, AA, AD, AE, NA, AN, PT, TP, AT or TA. In some embodiments, the
dipeptide is directly N-terminal to the amino acid sequences
defined in SEQ ID NOs: 1 to 76 and 110 (or the sequence variants
thereof defined herein). Such dipeptide sequence primarily serves
as a linker to connect the ankyrin repeat domain to the further
peptide sequence of the protein or as an extended alpha-helix of
the N-terminal capping module.
[0081] It is understood that for those embodiments of the
N-terminal capping module, which are defined by a certain amino
acid residue(s) in, e.g., position 15, as well as a minimal
sequence identity to an amino acid sequence or a defined number of
modifications compared to an amino acid sequence, both conditions
need to be fulfilled. For instance, an N-terminal capping module
having I in position 15 and at least 70% sequence identity to SEQ
ID NOs: 7 to 16, only relates to such embodiments wherein the
N-terminal capping module has I in position 15 and, at the same
time, at least 70% sequence identity to one or more of SEQ ID NOs:
7 to 16.
[0082] In some embodiments, the internal ankyrin repeats of the
ankyrin repeat domain consist of 33 amino acid residues.
[0083] In some embodiments, the internal ankyrin repeat(s) of the
ankyrin repeat domain of the invention comprise an amino acid
sequence that is substantially identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82 or from
the group consisting of SEQ ID NOs: 78 to 82. In some embodiments,
the internal ankyrin repeat(s) comprise an amino acid sequence that
has at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to 82
or from the group consisting of SEQ ID NOs: 78 to 82. In some
embodiments, the internal ankyrin repeat(s) comprise an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to 82
or from a variant of any one of SEQ ID NOs: 77 to 82 with up to 9,
up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up
to 1 modifications. In some embodiments, the internal ankyrin
repeat(s) comprise an amino acid sequence selected from the group
consisting of SEQ ID NOs: 78 to 82 or from a variant of any one of
SEQ ID NOs: 78 to 82 with up to 9, up to 8, up to 7, up to 6, up to
5, up to 4, up to 3, up to 2 or up to 1 modifications.
[0084] In some embodiments, the internal ankyrin repeat adjacent to
the N-terminal capping module of the ankyrin repeat domain of the
invention comprises an amino acid sequence that is substantially
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82 or from the group consisting of
SEQ ID NOs: 78 to 82. In some embodiments, the internal ankyrin
repeat adjacent to the N-terminal capping module comprises an amino
acid sequence that has at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or 100% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 77 to 82 or from the group consisting of SEQ ID NOs: 78 to 82.
In some embodiments, the internal ankyrin repeat adjacent to the
N-terminal capping module comprises an amino acid sequence selected
from the group consisting of SEQ ID NOs: 77 to 82 or from a variant
of any one of SEQ ID NOs: 77 to 82 with up to 9, up to 8, up to 7,
up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1
modifications. In some embodiments, the internal ankyrin repeat
adjacent to the N-terminal capping module comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 78 to 82
or from a variant of any one of SEQ ID NOs: 78 to 82 with up to 9,
up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up
to 1 modifications.
[0085] In some embodiments, the internal ankyrin repeats of an
ankyrin repeat domain of the invention share a high degree of
sequence identity or sequence similarity. In some embodiments, the
internal ankyrin repeat(s) share at least 70%, at least 75%, at
least 80%, at least 85%, at least 90% or at least 95% sequence
identity.
[0086] In some embodiments, the C-terminal capping module of the
ankyrin repeat domain of the invention comprises an amino acid
sequence that is substantially identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 83 to 92, 111 and
112. In some embodiments, the C-terminal capping module comprises
an amino acid sequence that has at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or 100%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 83 to 92, 111 and 112. In some
embodiments, the C-terminal capping module comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 83 to
92, 111 and 112 or from a variant of any one of SEQ ID NOs: 83 to
92, 111 and 112 with up to 9, up to 8, up to 7, up to 6, up to 5,
up to 4, up to 3, up to 2 or up to 1 modifications.
[0087] In some embodiments, the N-terminal capping module of the
ankyrin repeat domain of the invention comprises an amino acid
sequence that is substantially identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16 and the
internal ankyrin repeat(s) comprise an amino acid sequence that is
substantially identical to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 77 to 82. In some embodiments, the
N-terminal capping module comprises an amino acid sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16
and the internal ankyrin repeat(s) comprise an amino acid sequence
that has at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82. In some embodiments, the N-terminal capping module comprises an
amino acid sequence that has at least 70% sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino
acid sequence that has at least 70% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. In some embodiments, the N-terminal capping module comprises
an amino acid sequence that has at least 75% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino
acid sequence that has at least 75% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. In some embodiments, the N-terminal capping module comprises
an amino acid sequence that has at least 80% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino
acid sequence that has at least 80% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. In some embodiments, the N-terminal capping module comprises
an amino acid sequence that has at least 85% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino
acid sequence that has at least 85% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. In some embodiments, the N-terminal capping module comprises
an amino acid sequence that has at least 90% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino
acid sequence that has at least 90% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. In some embodiments, the N-terminal capping module comprises
an amino acid sequence that has at least 95% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino
acid sequence that has at least 95% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. In some embodiments, the N-terminal capping module comprises
an amino acid sequence that is identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16 and the
internal ankyrin repeat(s) comprise an amino acid sequence that is
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82.
[0088] In some embodiments, the N-terminal capping module comprises
an amino acid sequence that is substantially identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 7
to 16, the internal ankyrin repeat(s) comprise an amino acid
sequence that is substantially identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82, and the
C-terminal capping module comprises an amino acid sequence that is
substantially identical to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 83 to 92, 111 and 112. In some
embodiments, the N-terminal capping module comprises an amino acid
sequence that has at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or 100% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16, the internal ankyrin repeat(s) comprise an amino acid
sequence that has at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or 100% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 77 to 82, and the C-terminal capping module comprises an amino
acid sequence that has at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or 100% sequence identity to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 83 to 92, 111 and 112. In some embodiments, the N-terminal
capping module comprises an amino acid sequence that has at least
70% sequence identity to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 7 to 16, the internal ankyrin
repeat(s) comprise an amino acid sequence that has at least 70%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82, and the C-terminal capping
module comprises an amino acid sequence that has at least 70%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 83 to 92, 111 and 112. In some
embodiments, the N-terminal capping module comprises an amino acid
sequence that has at least 75% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16,
the internal ankyrin repeat(s) comprise an amino acid sequence that
has at least 75% sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82, and the
C-terminal capping module comprises an amino acid sequence that has
at least 75% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 83 to 92, 111 and 112. In
some embodiments, the N-terminal capping module comprises an amino
acid sequence that has at least 80% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 7
to 16, the internal ankyrin repeat(s) comprise an amino acid
sequence that has at least 80% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82, and the C-terminal capping module comprises an amino acid
sequence that has at least 80% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 83 to
92, 111 and 112. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that has at least 85% sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16, the internal ankyrin repeat(s)
comprise an amino acid sequence that has at least 85% sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82, and the C-terminal capping
module comprises an amino acid sequence that has at least 85%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 83 to 92, 111 and 112. In some
embodiments, the N-terminal capping module comprises an amino acid
sequence that has at least 90% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16,
the internal ankyrin repeat(s) comprise an amino acid sequence that
has at least 90% sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82, and the
C-terminal capping module comprises an amino acid sequence that has
at least 90% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 83 to 92, 111 and 112. In
some embodiments, the N-terminal capping module comprises an amino
acid sequence that has at least 95% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 7
to 16, the internal ankyrin repeat(s) comprise an amino acid
sequence that has at least 95% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82, and the C-terminal capping module comprises an amino acid
sequence that has at least 95% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 83 to
92, 111 and 112. In some embodiments, the N-terminal capping module
comprises an amino acid sequence that is identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16,
the internal ankyrin repeat(s) comprise an amino acid sequence that
is identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82, and the C-terminal capping
module comprises an amino acid sequence that is identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 83 to 92, 111 and 112.
[0089] In some embodiments, the N-terminal capping module comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 or from a variant of any one of SEQ ID NOs: 7 to 16
with up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3,
up to 2 or up to 1 modifications outside position 15, and wherein X
at position 15 of SEQ ID NOs: 7 to 16 or said variants thereof is
an amino acid residue as defined above for position 15 and the
internal ankyrin repeat(s) comprise an amino acid sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to 82
or SEQ ID NOs: 78 to 82.
[0090] In some embodiments, the N-terminal capping module comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 or from a variant of any one of SEQ ID NOs: 7 to 16
with up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3,
up to 2 or up to 1 modifications outside position 15, and wherein X
at position 15 of SEQ ID NOs: 7 to 16 or said variants thereof is
an amino acid residue as defined above for position 15, the
internal ankyrin repeat(s) comprise an amino acid sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, or 100% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to 82
or SEQ ID NOs: 78 to 82, and the C-terminal capping module
comprises an amino acid sequence that has at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, or
100% sequence identity to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 83 to 92, 111 and 112.
[0091] In some embodiments, the ankyrin repeat domain comprises the
N-terminal capping module, one internal ankyrin repeat and a
C-terminal capping module (N1C). Such ankyrin repeat domains are
shown in Example 1. In some embodiments, the ankyrin repeat domain
comprises the N-terminal capping module, multiple internal ankyrin
repeats and a C-terminal capping module. In one embodiment, the
ankyrin repeat domain comprises the N-terminal capping module, 2 or
3 internal ankyrin repeats and a C-terminal capping module (N2C or
N3C, respectively). In one embodiment, the ankyrin repeat domain is
N2C. In another embodiment, the ankyrin repeat domain is N3C.
[0092] In some embodiments, the protein of the invention is a
recombinant protein or DARPin.
[0093] In some embodiments, the ankyrin repeat domain of the
protein of the invention specifically binds to a target. For
instance, the ankyrin repeat domain may specifically bind to a
mammalian serum albumin, such as human serum albumin.
[0094] The protein of the invention may comprise one or more
further moieties in addition to the ankyrin repeat domain
comprising the N-terminal capping module, such as a moiety binding
to a target, a labeling moiety, a toxic moiety, a moiety improving
the pharmacokinetics, a moiety providing effector functions, a
moiety allowing for the purification of the protein or a moiety
providing enzymatic activity. In some embodiments, the further
moiety binding to a target is another ankyrin repeat domain, an
antibody or fragment thereof or a receptor protein. In some
embodiments, the further moiety binding to a target is another
ankyrin repeat domain. In some embodiments, the labeling moiety is
a stable isotope, a mass tag or a fluorescent label. In some
embodiments, the toxic moiety is a chemotherapeutic agent, such as
an alkylating agent, an antimetabolite, a taxane, or an
anthracycline. In some embodiments, the moiety improving
pharmacokinetics is a pegylation, a mammalian serum albumin, an
immunoglobulin, a Fc domain of an immunoglobulin or a moiety
binding to mammalian serum albumin or to an immunoglobulin. In one
embodiment, the protein further contains an ankyrin repeat domain
binding to a mammalian serum albumin. In some embodiments, the
further moiety providing effector functions is a Fc domain of an
immunoglobulin. In some embodiments, the moiety allowing for the
purification of the protein is a FLAG-tag, a GST-tag, an HA-tag, a
Myc-tag, a His-tag or a Strep-tag. In some embodiments, the further
moiety providing enzymatic or fluorescence activity is, e.g.,
beta-lactamase or green fluorescence protein, respectively.
[0095] The further moiety may be proteinaceous or
non-proteinaceous.
[0096] In some embodiments, the further moiety in addition to the
ankyrin repeat domain comprising the N-terminal capping module is
one or more additional ankyrin repeat domains. In some embodiments,
the protein of the invention comprises more than one, e.g., at
least two, at least three, at least four or at least five, ankyrin
repeat domains. In some embodiments, the protein of the invention
comprises more than one, e.g., at least two, at least three, at
least four or at least five, ankyrin repeat domains each comprising
the N-terminal capping module of the invention. In some
embodiments, the protein of the invention comprises multiple
ankyrin repeat domains which specifically bind to multiple targets.
In some embodiments, the protein of the invention comprises a
single ankyrin repeat domain.
[0097] The further moiety may connect covalently to the protein,
for instance, via a peptide linker or via a maleimide-containing
crosslinker. Suitable peptide linkers include glycine-serine
linkers and proline-threonine linkers. In some embodiments, the
peptide linkers have a length of 2 to 24 amino acid residues or 2
to 16 amino acid residues. Alternatively, the further moiety may
connect non-covalently to the protein, for instance, via a
multimerization moiety. In some embodiments, a multimerization
moiety is an immunoglobulin heavy chain constant region, a leucine
zipper or a free thiol which can form a disulfide bond with another
free thiol.
[0098] In a further aspect, the present disclosure relates to a
protein library of the proteins of the invention. At least two of
these proteins of the protein library differ in the amino acid
sequence of their ankyrin repeat domains. In some embodiments, the
protein library has at least 10.sup.3, at least 10.sup.5, at least
10.sup.7, least 10.sup.9, least 10.sup.11 or at least 10.sup.13
proteins that differ in the amino acid sequence of the ankyrin
repeat domain.
[0099] In some embodiments, the protein library comprises proteins
of the invention having different ankyrin repeat domain structures.
For instance, the protein library may contain a mixture of proteins
with proteins having the N-terminal capping module, 2 internal
ankyrin repeats and a C-terminal capping module and proteins having
the N-terminal capping module, 3 internal ankyrin repeats and a
C-terminal capping module. In some embodiments, the proteins of the
protein library all share the same ankyrin repeat domain structure.
For instance, the ankyrin repeat domain of all proteins of the
library comprises the N-terminal capping module, 2 internal ankyrin
repeats and a C-terminal capping module. In other embodiments, the
ankyrin repeat domain of all proteins of the library comprises the
N-terminal capping module, 3 internal ankyrin repeats and a
C-terminal capping module. In some embodiments, the proteins of the
library each comprise a single ankyrin repeat domain only.
[0100] The sequence variability in the ankyrin repeat domains of
the protein library may be brought about randomly, e.g., by
error-prone PCR of the nucleic acid molecules encoding the
proteins, or it may be obtained by rational design followed by,
e.g., direct synthesis of the nucleic acid molecules encoding the
proteins ("design approach"). In some embodiments, the variability
is introduced by the design approach. In the design approach,
variability of the amino acid sequence is introduced in one or more
than one position of the ankyrin repeat domains, which variable
positions are also referred to as "randomized positions", i.e.
those positions that can potentially be occupied by more than one
amino acid residue, whereas the remaining positions remain
unchanged and are also referred to as "fixed positions", i.e. those
positions that are occupied by a specific amino acid residue. In
some embodiments, the randomized positions are those positions
occupied by potential target interaction residues and/or the fixed
positions are those positions occupied by framework residues. In
some embodiments, a subset of the positions occupied by potential
target interaction residues are randomized positions. In some
embodiments, all positions occupied by potential target interaction
residues are randomized positions.
[0101] In certain embodiments, there are corresponding fixed
positions and randomized positions in the different proteins of the
protein library. Due to the intended variability in the randomized
positions, the amino acid residues in each corresponding randomized
position will usually differ, although there may also be identical
amino acid residues in corresponding randomized positions for at
least some of the proteins in the library (though, in such cases,
the proteins will not necessarily have identical amino acid
residues in each of their corresponding randomized positions). In
some embodiments, the fixed positions and the randomized positions
are the same for the ankyrin repeat domains of each protein of the
protein library. In some embodiments wherein the ankyrin repeat
domains have multiple internal ankyrin repeats, the internal
ankyrin repeats of each ankyrin repeat domain have different
randomized and fixed positions. In some embodiments wherein the
ankyrin repeat domains have multiple internal ankyrin repeats, the
internal ankyrin repeats of each ankyrin repeat domain have the
same randomized and fixed positions.
[0102] The randomized positions may show different degrees of
variability, i.e. they may potentially be occupied by a different
set of amino acid residues. In some embodiments, the degree of
variability differs between randomized positions. In some
embodiments, the amino acid residue in a randomized position is any
of the twenty natural amino acid residues. In some embodiments, a
randomized position may only be occupied by a subset of the twenty
natural amino acid residues. Such subsets can be those having
common physicochemical properties, such as sets of hydrophobic,
hydrophilic, acidic, basic, aromatic, or aliphatic amino acid
residues. Other subsets are those comprising all twenty natural
amino acid residues except for certain non-desired amino acid
residues, such as sets not comprising cysteines or prolines. In yet
other embodiments, the subsets comprise those amino acid residues
that are found in the corresponding positions of naturally
occurring ankyrin repeats.
[0103] In some embodiments, the proteins of the protein library
share at least 70% sequence identity, at least 80% sequence
identity, or at least 90% sequence identity in the amino acid
sequence of their ankyrin repeat domains.
[0104] The above protein library can serve to select those proteins
of the library that have a predetermined property, i.e. a certain
property of interest that may be found in one of the proteins of
the protein library and that can be screened for. Such
predetermined property may include the specific binding to a
target, the activation or inhibition of a target, such as an
enzyme, and the blocking of an interaction between two targets. In
some embodiments, the predetermined property is the specific
binding to a target.
[0105] In one embodiment, the present disclosure provides a method
for selecting a protein having a predetermined property, comprising
the following steps: [0106] a) Providing a protein library of said
proteins comprising an ankyrin repeat domain with the N-terminal
capping module; [0107] b) Selecting a protein from the library
having the predetermined property.
[0108] During the selection step b), the proteins can be selected
using screening methods commonly known to the person skilled in the
art, such as yeast display, protein fragment complementation assay,
phage display or ribosome display. In some embodiments, the
proteins are selected in step b) using phage display or ribosome
display.
[0109] After the selection of a protein, the protein can be further
modified, mutated and/or optimized by methods commonly known in the
art.
[0110] For instance, amino acid sequence variants of the protein
can be generated, e.g., by subjecting the nucleic acid encoding the
selected protein to physical or chemical mutagens, copying said
nucleic acid by error-prone PCR, using said nucleic acid for DNA
shuffling or random chimeragenesis (Neylon C., Nucleic Acids Res.,
32(4), 1448-1459, 2004). The protein library of such amino acid
sequence variants may then again be subjected to the above
selection step b) in order to select the variant(s) having the
predetermined property.
[0111] The protein selected in step b) above may also be
selectively mutated. For instance, one or more cysteine residues
may be introduced, the thiol group(s) of which can then react with
maleimide cross-linkers. Similarly, certain non-desirable amino
acid residues may be removed, for instance, cysteines, which are
prone to oxidations. Also, amino acid residues may be selectively
mutated after analysis of the crystal structure so that the protein
structure better fits to the target.
[0112] The protein selected in step b) may also become modified
with the one or more further moieties in addition to the ankyrin
repeat domain outlined above for the protein of the invention.
[0113] In further aspects, the present disclosure provides a
nucleic acid encoding the protein of the invention, a vector
comprising such nucleic acid and a cell or in vitro expression
system comprising such nucleic acid or such vector.
[0114] The nucleic acid can be DNA or RNA, single stranded or
double-stranded, in isolated form or part of a larger nucleic acid,
e.g., of a vector or a chromosome. The nucleic acid may comprise
elements that enable delivery of the nucleic acid to a cell and/or
expression of the nucleic acid in a cell. For instance, the nucleic
acid encoding the protein of the invention can be operatively
linked to expression control sequences, which have an impact on the
transcription and/or translation of the protein, such as promoters,
enhancers, transcription terminators, start codons and stop codons.
Depending on the intended application and/or context, the
expression control sequences may be selected from any eukaryotic or
prokaryotic organism. Suitable promoters may be constitutive or
inducible promoters. Examples include the CMV-, lacZ-, T7-, T5-,
RSV-, SV40-, AOX1-, and GAPDH-promoter. Suitable enhancers include
the CMV-enhancer, insulin-responsive elements, and an
SV40-enhancer. Transcription terminators include the SV40-, lacZ-,
and tk-polyadenylation signal.
[0115] The present disclosure also provides a library with nucleic
acid molecules encoding the protein of the invention. At least two
of the nucleic acid molecules of the nucleic acid library differ in
the nucleic acid sequence of their ankyrin repeat domains. In some
embodiments, the nucleic acid library has at least 10.sup.3, at
least 10.sup.5, at least 10.sup.7, or at least 10.sup.9 nucleic
acid molecules that differ in the nucleic acid sequence coding for
the ankyrin repeat domain.
[0116] The cell comprising the nucleic acid or vector encoding the
protein of the invention can be a prokaryotic or a eukaryotic cell.
In some embodiments, the cell is a bacterial, yeast or mammalian
cell. In some embodiments, the cell is derived from E. coli, P.
pastoris, S. cerevisiae, human, hamster or mouse. In some
embodiments, the cell is selected from CHO, HEK293, BHK, NS0,
Sp2/0, HT-1080, PER.C6, CAP and HuH-7 cells.
[0117] In some embodiments, the in vitro expression system
comprising the nucleic acid or vector encoding the protein of the
invention is based on a cell-free extract from E. coli, yeast,
rabbit, wheat germ, insect or human.
[0118] In a further aspect, the present disclosure provides a
method of preparing a protein comprising the following steps:
[0119] a) culturing a cell comprising a nucleic acid encoding the
protein of the invention under conditions allowing expression
thereof; and [0120] b) purifying the expressed protein.
[0121] In one embodiment, the present disclosure provides a method
of modifying an existing ankyrin repeat domain by replacing the
N-terminal capping module of the existing ankyrin repeat domain by
an N-terminal capping module disclosed herein. By modifying an
existing ankyrin repeat domain in this way, the favorable
properties related to the N-terminal capping module disclosed
herein may be transferred to the existing ankyrin repeat
domain.
[0122] Thus, in one embodiment, the present disclosure provides a
method of preparing a protein comprising an ankyrin repeat domain
with an improved thermostability, such as a higher melting
temperature and/or higher fraction of refolded ankyrin repeat
domains after thermal denaturation, comprising the following steps:
[0123] a) selecting a protein comprising an ankyrin repeat domain;
[0124] b) determining the amino acid sequence of the N-terminal
capping module of said ankyrin repeat domain; [0125] c)
substituting the amino acid residue in the position of the
N-terminal capping module corresponding to position 15 of SEQ ID
NO: 3 by an amino acid residue disclosed for position 15 for the
protein of the invention herein; and [0126] wherein the
thermostability of the resulting ankyrin repeat protein is improved
in comparison to a reference ankyrin repeat domain having the same
amino acid sequence except for the mutation(s) in position 15 of
the N-terminal capping module.
[0127] For instance, the amino acid residue corresponding to
position 15 of SEQ ID NO: 3 may be substituted by an amino acid
residue selected from the group consisting of I, T, A, V, L and
M.
[0128] Thus, in one embodiment, the present disclosure provides a
method of preparing a protein comprising an ankyrin repeat domain
with an improved thermostability, such as a higher melting
temperature and/or higher fraction of refolded ankyrin repeat
domains after thermal denaturation, comprising the following steps:
[0129] a) selecting a protein comprising an ankyrin repeat domain
having an amino acid residue which is none of I, T, A, V, L and M
at the position of the N-terminal capping module corresponding to
position 15 of SEQ ID NO: 3; [0130] b) substituting the amino acid
residue in said position by an amino acid residue selected from the
group consisting of I, T, A, V, L and M.
[0131] In one embodiment, the present disclosure provides a method
of preparing a protein comprising the following steps: [0132] a)
assembling by genetic means one or more gene(s) encoding the
protein of the invention, wherein one gene comprises sequence
encoding the ankyrin repeat domain that comprises the N-terminal
capping module, followed by one or more internal ankyrin repeats
and a C-terminal capping module, and [0133] b) expressing the
gene(s) encoding the protein of the invention.
[0134] The present disclosure also provides a pharmaceutical
composition comprising the protein of the invention. In some
embodiments, the pharmaceutical composition comprises the protein
of the invention and a pharmaceutically acceptable carrier.
[0135] In a further aspect, the present disclosure provides the use
of the proteins of the invention in a method of treatment.
Further Embodiments
[0136] 1. A protein comprising an ankyrin repeat domain with an
N-terminal capping module, wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of I, T, A, V, L, M, S,
Q and R. [0137] 2. A protein comprising an ankyrin repeat domain
with an N-terminal capping module, wherein the amino acid residue
of said N-terminal capping module corresponding to position 15 in
SEQ ID NO: 3 is selected from the group consisting of I, T, A, V,
L, M and S. [0138] 3. A protein comprising an ankyrin repeat domain
with an N-terminal capping module, wherein the amino acid residue
of said N-terminal capping module corresponding to position 15 in
SEQ ID NO: 3 is selected from the group consisting of I, T, A, V, L
and M. [0139] 4. A protein comprising an ankyrin repeat domain with
an N-terminal capping module, wherein the amino acid residue of
said N-terminal capping module corresponding to position 15 in SEQ
ID NO: 3 is selected from the group consisting of I, T, A, V, L and
M and the amino acid residue of said N-terminal capping module
corresponding to position 22 in SEQ ID NO: 3 is selected from the
group consisting of L, I and V. [0140] 5. The protein according to
any one of items 1 to 4, wherein said N-terminal capping module
comprises an amino acid sequence that is substantially identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16. [0141] 6. The protein according to any one of items 1
to 4, wherein said N-terminal capping module comprises an amino
acid sequence that has at least 70% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 7
to 16. [0142] 7. The protein according to any one of items 1 to 4,
wherein said N-terminal capping module comprises an amino acid
sequence that has at least 75% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16.
[0143] 8. The protein according to any one of items 1 to 4, wherein
said N-terminal capping module comprises an amino acid sequence
that has at least 80% sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16. [0144]
9. The protein according to any one of items 1 to 4, wherein said
N-terminal capping module comprises an amino acid sequence that has
at least 85% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16. [0145] 10. The
protein according to any one of items 1 to 4, wherein said
N-terminal capping module comprises an amino acid sequence that has
at least 90% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16. [0146] 11. The
protein according to any one of items 1 to 4, wherein said
N-terminal capping module comprises an amino acid sequence that has
at least 95% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16. [0147] 12. The
protein according to any one of items 1 to 4, wherein said
N-terminal capping module comprises an amino acid sequence that is
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 76 and 110. [0148] 13. A protein
comprising an ankyrin repeat domain, [0149] wherein said ankyrin
repeat domain comprises an N-terminal capping module having an
amino acid sequence that has at least 70% sequence identity with an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16; and [0150] wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of I, T, A, V, L and M.
[0151] 14. A protein comprising an ankyrin repeat domain, [0152]
wherein said ankyrin repeat domain comprises an N-terminal capping
module having an amino acid sequence that has at least 75% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16; and [0153] wherein the amino
acid residue of said N-terminal capping module corresponding to
position 15 in SEQ ID NO: 3 is selected from the group consisting
of I, T, A, V, L and M. [0154] 15. A protein comprising an ankyrin
repeat domain, [0155] wherein said ankyrin repeat domain comprises
an N-terminal capping module having an amino acid sequence that has
at least 80% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16; and [0156]
wherein the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of I, T, A, V, L and M. [0157] 16. A protein
comprising an ankyrin repeat domain, [0158] wherein said ankyrin
repeat domain comprises an N-terminal capping module having an
amino acid sequence that has at least 85% sequence identity with an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16; and [0159] wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of I, T, A, V, L and M.
[0160] 17. A protein comprising an ankyrin repeat domain, [0161]
wherein said ankyrin repeat domain comprises an N-terminal capping
module having an amino acid sequence that has at least 90% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16; and [0162] wherein the amino
acid residue of said N-terminal capping module corresponding to
position 15 in SEQ ID NO: 3 is selected from the group consisting
of I, T, A, V, L and M. [0163] 18. A protein comprising an ankyrin
repeat domain, [0164] wherein said ankyrin repeat domain comprises
an N-terminal capping module having an amino acid sequence that has
at least 95% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16; and [0165]
wherein the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of I, T, A, V, L and M. [0166] 19. A protein
comprising an ankyrin repeat domain, [0167] wherein said ankyrin
repeat domain comprises an N-terminal capping module that has an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 or from a variant of any one of SEQ ID NOs: 7 to 16
with up to 9 modifications outside position 15; and [0168] wherein
X at position 15 in SEQ ID NOs: 7 to 16 or said variants thereof is
an amino acid residue selected from the group consisting of I, T,
A, V, L and M. [0169] 20. A protein comprising an ankyrin repeat
domain, [0170] wherein said ankyrin repeat domain comprises an
N-terminal capping module that has an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16 or from a variant
of any one of SEQ ID NOs: 7 to 16 with up to 8 modifications
outside position 15; and [0171] wherein X at position 15 in SEQ ID
NOs: 7 to 16 or said variants thereof is an amino acid residue
selected from the group consisting of I, T, A, V, L and M. [0172]
21. A protein comprising an ankyrin repeat domain, [0173] wherein
said ankyrin repeat domain comprises an N-terminal capping module
that has an amino acid sequence selected from the group consisting
of SEQ ID NOs: 7 to 16 or from a variant of any one of SEQ ID NOs:
7 to 16 with up to 6 modifications outside position 15; and [0174]
wherein X at position 15 in SEQ ID NOs: 7 to 16 or said variants
thereof is an amino acid residue selected from the group consisting
of I, T, A, V, L and M. [0175] 22. A protein comprising an ankyrin
repeat domain, [0176] wherein said ankyrin repeat domain comprises
an N-terminal capping module that has an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16 or from a
variant of any one of SEQ ID NOs: 7 to 16 with up to 4
modifications outside position 15; and [0177] wherein X at position
15 in SEQ ID NOs: 7 to 16 or said variants thereof is an amino acid
residue selected from the group consisting of I, T, A, V, L and M.
[0178] 23. A protein comprising an ankyrin repeat domain, [0179]
wherein said ankyrin repeat domain comprises an N-terminal capping
module that has an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16 or from a variant of any one of
SEQ ID NOs: 7 to 16 with up to 2 modifications outside position 15;
and [0180] wherein X at position 15 in SEQ ID NOs: 7 to 16 or said
variants thereof is an amino acid residue selected from the group
consisting of I, T, A, V, L and M. [0181] 24. A protein comprising
an ankyrin repeat domain, [0182] wherein said ankyrin repeat domain
comprises an N-terminal capping module that has an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16
or from a variant of any one of SEQ ID NOs: 7 to 16 with up to 1
modification outside position 15; and [0183] wherein X at position
15 in SEQ ID NOs: 7 to 16 or said variants thereof is an amino acid
residue selected from the group consisting of I, T, A, V, L and M.
[0184] 25. The protein according to any one of items 19 to 22,
wherein the combined number of deletions and insertions of said
variant of any one of SEQ ID NOs: 7 to 16 is not more than 3.
[0185] 26. The protein according to any one of items 19 to 22,
wherein the combined number of deletions and insertions of said
variant of any one of SEQ ID NOs: 7 to 16 is not more than 2.
[0186] 27. The protein according to any one of items 19 to 22,
wherein the combined number of deletions and insertions of said
variant of any one of SEQ ID NOs: 7 to 16 is not more than 1.
[0187] 28. A protein comprising an ankyrin repeat domain, [0188]
wherein said ankyrin repeat domain comprises an N-terminal capping
module that has an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16 or from a variant of any one of
SEQ ID NOs: 7 to 16 with up to 9 substitutions in other positions
than position 15; and [0189] wherein X at position 15 in SEQ ID
NOs: 7 to 16 or said variants thereof is an amino acid residue
selected from the group consisting of I, T, A, V, L and M. [0190]
29. A protein comprising an ankyrin repeat domain, [0191] wherein
said ankyrin repeat domain comprises an N-terminal capping module
that has an amino acid sequence selected from the group consisting
of SEQ ID NOs: 7 to 16 or from a variant of any one of SEQ ID NOs:
7 to 16 with up to 8 substitutions in other positions than position
15; and [0192] wherein X at position 15 in SEQ ID NOs: 7 to 16 or
said variants thereof is an amino acid residue selected from the
group consisting of I, T, A, V, L and M. [0193] 30. A protein
comprising an ankyrin repeat domain, [0194] wherein said ankyrin
repeat domain comprises an N-terminal capping module that has an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16 or from a variant of any one of SEQ ID NOs: 7 to 16
with up to 6 substitutions in other positions than position 15; and
[0195] wherein X at position 15 in SEQ ID NOs: 7 to 16 or said
variants thereof is an amino acid residue selected from the group
consisting of I, T, A, V, L and M. [0196] 31. A protein comprising
an ankyrin repeat domain, [0197] wherein said ankyrin repeat domain
comprises an N-terminal capping module that has an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 to 16
or from a variant of any one of SEQ ID NOs: 7 to 16 with up to 4
substitutions in other positions than position 15; and [0198]
wherein X at position 15 in SEQ ID NOs: 7 to 16 or said variants
thereof is an amino acid residue selected from the group consisting
of I, T, A, V, L and M. [0199] 32. A protein comprising an ankyrin
repeat domain, [0200] wherein said ankyrin repeat domain comprises
an N-terminal capping module that has an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16 or from a
variant of any one of SEQ ID NOs: 7 to 16 with up to 2
substitutions in other positions than position 15; and [0201]
wherein X at position 15 in SEQ ID NOs: 7 to 16 or said variants
thereof is an amino acid residue selected from the group consisting
of I, T, A, V, L and M. [0202] 33. A protein comprising an ankyrin
repeat domain, [0203] wherein said ankyrin repeat domain comprises
an N-terminal capping module that has an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16 or from a
variant of any one of SEQ ID NOs: 7 to 16 with up to 1 substitution
in other positions than position 15; and [0204] wherein X at
position 15 in SEQ ID NOs: 7 to 16 or said variants thereof is an
amino acid residue selected from the group consisting of I, T, A,
V, L and M. [0205] 34. A protein comprising an ankyrin repeat
domain, wherein said ankyrin repeat domain comprises an N-terminal
capping module having an amino acid sequence that has at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or 100% sequence identity with SEQ ID NO: 58, and [0206]
wherein the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of I, T, A, V, L and M. [0207] 35. The protein
according to any one of items 1 to 34, wherein said N-terminal
capping module further has an amino acid residue selected from the
group consisting of G, A, D and S at the position corresponding to
position 3 in SEQ ID NO: 3. [0208] 36. The protein according to
item 35, wherein said amino acid residue of the N-terminal capping
module corresponding to position 3 in SEQ ID NO: 3 is A or S.
[0209] 37. The protein according to any one of items 1 to 36,
wherein said N-terminal capping module further has an amino acid
residue selected from the group consisting of L, S, Q, K, R, A, H,
D and E at the position corresponding to position 11 in SEQ ID NO:
3. [0210] 38. The protein according to item 37, wherein said amino
acid residue of the N-terminal capping module corresponding to
position 11 in SEQ ID NO: 3 is selected from the group consisting
of K, E, Q, A and L. [0211] 39. The protein according to any one of
items 1 to 38, wherein said N-terminal capping module further has
an amino acid residue selected from the group consisting of D, A,
N, Q, G, S, T, K, E and R at the position corresponding to position
17 in SEQ ID NO: 3. [0212] 40. The protein according to item 39,
wherein said amino acid residue of the N-terminal capping module
corresponding to position 17 in SEQ ID NO: 3 is A. [0213] 41. The
protein according to any one of items 1 to 40, wherein said
N-terminal capping module further has an amino acid residue
selected from the group consisting of N, K, R, T, S, E, Q, A, D and
H at the position corresponding to position 20 in SEQ ID NO: 3.
[0214] 42. The protein according to item 41, wherein said amino
acid residue of the N-terminal capping module corresponding to
position 20 in SEQ ID NO: 3 is Q.
[0215] 43. A protein comprising an ankyrin repeat domain with an
N-terminal capping module, wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of I, T, A, V, L and M,
the amino acid residue of said N-terminal capping module
corresponding to position 3 in SEQ ID NO: 3 is A or S, the amino
acid residue of said N-terminal capping module corresponding to
position 11 in SEQ ID NO: 3 is selected from the group consisting
of K, E, Q, A and L, the amino acid residue of said N-terminal
capping module corresponding to position 17 in SEQ ID NO: 3 is A
and the amino acid residue of said N-terminal capping module
corresponding to position 20 in SEQ ID NO: 3 is Q. [0216] 44. The
protein according to any one of items 1 to 43, wherein said
N-terminal capping module further has the amino acid residues G and
S or D and A N-terminal to the amino acid sequence corresponding to
SEQ ID NO: 3 at the positions corresponding to -2 and -1 of SEQ ID
NO: 3, respectively. [0217] 45. The protein according to any one of
items 1 to 44, wherein said N-terminal capping module is an
N-terminal capping repeat. [0218] 46. The protein according to any
one of items 1 to 45, wherein said ankyrin repeat domain has one or
more internal ankyrin repeats, each consisting of 33 amino acid
residues. [0219] 47. The protein according to any one of items 1 to
46, wherein said ankyrin repeat domain has two or three internal
ankyrin repeats. [0220] 48. The protein according to any one of
items 1 to 47, wherein said ankyrin repeat domain comprises one or
more internal ankyrin repeats, and [0221] wherein said one or more
internal ankyrin repeats each have an amino acid sequence having at
least 70% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 77 to 82. [0222] 49. The
protein according to any one of items 1 to 47, wherein said ankyrin
repeat domain comprises one or more internal ankyrin repeats, and
[0223] wherein said one or more internal ankyrin repeats each have
an amino acid sequence that is substantially identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 77
to 82. [0224] 50. The protein according to any one of items 1 to
49, wherein said ankyrin repeat domain comprises one or more
internal ankyrin repeats, and [0225] wherein said one or more
internal ankyrin repeats each comprise an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82 or from
a variant of any one of SEQ ID NOs: 77 to 82 with up to 9, up to 8,
up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1
modifications. [0226] 51. The protein according to any one of items
1 to 49, wherein the internal ankyrin repeat of the ankyrin repeat
domain that is adjacent to said N-terminal capping module comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 77 to 82 or from a variant of any one of SEQ ID NOs: 77 to 82
with up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3,
up to 2 or up to 1 modifications. [0227] 52. The protein according
to any one of items 1 to 51, wherein said ankyrin repeat domain
comprises more than one internal ankyrin repeat, and [0228] wherein
said internal ankyrin repeats share at least 70% sequence identity.
[0229] 53. The protein according to any one of items 1 to 52,
wherein said ankyrin repeat domain further comprises a C-terminal
capping module having at least 70% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID NOs: 83
to 92, 111 and 112. [0230] 54. The protein according to any one of
items 1 to 52, wherein said ankyrin repeat domain further comprises
a C-terminal capping module having an amino acid sequence that is
substantially identical to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 83 to 92, 111 and 112. [0231] 55. A
protein comprising an ankyrin repeat domain, wherein said ankyrin
repeat domain comprises an N-terminal capping module, one or more
internal ankyrin repeats and a C-terminal capping module, [0232]
wherein said N-terminal capping module has an amino acid sequence
that has at least 70% sequence identity with an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16, [0233]
wherein the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of L, M, I, T, A, V and S, and [0234] wherein said
one or more internal ankyrin repeats each have an amino acid
sequence that has at least 70% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82. [0235] 56. A protein comprising an ankyrin repeat domain,
wherein said ankyrin repeat domain comprises an N-terminal capping
module, one or more internal ankyrin repeats and a C-terminal
capping module, [0236] wherein said N-terminal capping module has
an amino acid sequence that has at least 75% sequence identity with
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16, [0237] wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of L, M, I, T, A, V and
S, and [0238] wherein said one or more internal ankyrin repeats
each have an amino acid sequence that has at least 75% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82. [0239] 57. A protein comprising
an ankyrin repeat domain, wherein said ankyrin repeat domain
comprises an N-terminal capping module, one or more internal
ankyrin repeats and a C-terminal capping module, [0240] wherein
said N-terminal capping module has an amino acid sequence that has
at least 80% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16, [0241] wherein
the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of L, M, I, T, A, V and S, and [0242] wherein said
one or more internal ankyrin repeats each have an amino acid
sequence that has at least 80% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82. [0243] 58. A protein comprising an ankyrin repeat domain,
wherein said ankyrin repeat domain comprises an N-terminal capping
module, one or more internal ankyrin repeats and a C-terminal
capping module, [0244] wherein said N-terminal capping module has
an amino acid sequence that has at least 85% sequence identity with
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16, [0245] wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of L, M, I, T, A, V and
S, and [0246] wherein said one or more internal ankyrin repeats
each have an amino acid sequence that has at least 85% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82. [0247] 59. A protein comprising
an ankyrin repeat domain, wherein said ankyrin repeat domain
comprises an N-terminal capping module, one or more internal
ankyrin repeats and a C-terminal capping module, [0248] wherein
said N-terminal capping module has an amino acid sequence that has
at least 90% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16, [0249] wherein
the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of L, M, I, T, A, V and S, and [0250] wherein said
one or more internal ankyrin repeats each have an amino acid
sequence that has at least 90% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82. [0251] 60. A protein comprising an ankyrin repeat domain,
wherein said ankyrin repeat domain comprises an N-terminal capping
module, one or more internal ankyrin repeats and a C-terminal
capping module, [0252] wherein said N-terminal capping module has
an amino acid sequence that has at least 95% sequence identity with
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16, [0253] wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of L, M, I, T, A, V and
S, and [0254] wherein said one or more internal ankyrin repeats
each have an amino acid sequence that has at least 95% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82. [0255] 61. A protein comprising
an ankyrin repeat domain, wherein said ankyrin repeat domain
comprises an N-terminal capping module, one or more internal
ankyrin repeats and a C-terminal capping module, [0256] wherein
said N-terminal capping module has an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16, [0257] wherein
the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of L, M, I, T, A, V and S, and [0258] wherein said
one or more internal ankyrin repeats each have an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82. [0259] 62. A protein comprising an ankyrin repeat domain,
wherein said ankyrin repeat domain comprises an N-terminal capping
module, one or more internal ankyrin repeats and a C-terminal
capping module, [0260] wherein said N-terminal capping module has
an amino acid sequence that has at least 70% sequence identity with
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 7 to 16, [0261] wherein the amino acid residue of said
N-terminal capping module corresponding to position 15 in SEQ ID
NO: 3 is selected from the group consisting of L, M, I, T, A, V and
S, [0262] wherein said one or more internal ankyrin repeats each
have an amino acid sequence that has at least 70% sequence identity
with an amino acid sequence selected from the group consisting of
SEQ ID NOs: 77 to 82, and [0263] wherein said C-terminal capping
module has an amino acid sequence that has at least 70% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 83 to 92, 111 and 112. [0264] 63. A
protein comprising an ankyrin repeat domain, wherein said ankyrin
repeat domain comprises an N-terminal capping module, one or more
internal ankyrin repeats and a C-terminal capping module, [0265]
wherein said N-terminal capping module has an amino acid sequence
that has at least 75% sequence identity with an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7 to 16, [0266]
wherein the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of L, M, I, T, A, V and S, [0267] wherein said one
or more internal ankyrin repeats each have an amino acid sequence
that has at least 75% sequence identity with an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82, and
[0268] wherein said C-terminal capping module has an amino acid
sequence that has at least 75% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 83 to
92, 111 and 112. [0269] 64. A protein comprising an ankyrin repeat
domain, wherein said ankyrin repeat domain comprises an N-terminal
capping module, one or more internal ankyrin repeats and a
C-terminal capping module, [0270] wherein said N-terminal capping
module has an amino acid sequence that has at least 80% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16, [0271] wherein the amino acid
residue of said N-terminal capping module corresponding to position
15 in SEQ ID NO: 3 is selected from the group consisting of L, M,
I, T, A, V and S, [0272] wherein said one or more internal ankyrin
repeats each have an amino acid sequence that has at least 80%
sequence identity with an amino acid sequence selected from the
group consisting of SEQ ID NOs: 77 to 82, and [0273] wherein said
C-terminal capping module has an amino acid sequence that has at
least 80% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 83 to 92, 111 and 112.
[0274] 65. A protein comprising an ankyrin repeat domain, wherein
said ankyrin repeat domain comprises an N-terminal capping module,
one or more internal ankyrin repeats and a C-terminal capping
module, [0275] wherein said N-terminal capping module has an amino
acid sequence that has at least 85% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID NOs: 7
to 16, [0276] wherein the amino acid residue of said N-terminal
capping module corresponding to position 15 in SEQ ID NO: 3 is
selected from the group consisting of L, M, I, T, A, V and S,
[0277] wherein said one or more internal ankyrin repeats each have
an amino acid sequence that has at least 85% sequence identity with
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 77 to 82, and [0278] wherein said C-terminal capping module
has an amino acid sequence that has at least 85% sequence identity
with an amino acid sequence selected from the group consisting of
SEQ ID NOs: 83 to 92, 111 and 112. [0279] 66. A protein comprising
an ankyrin repeat domain, wherein said ankyrin repeat domain
comprises an N-terminal capping module, one or more internal
ankyrin repeats and a C-terminal capping module, [0280] wherein
said N-terminal capping module has an amino acid sequence that has
at least 90% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 16, [0281] wherein
the amino acid residue of said N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3 is selected from the
group consisting of L, M, I, T, A, V and S, [0282] wherein said one
or more internal ankyrin repeats each have an amino acid sequence
that has at least 90% sequence identity with an amino acid sequence
selected from the group consisting of SEQ ID NOs: 77 to 82, and
[0283] wherein said C-terminal capping module has an amino acid
sequence that has at least 90% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 83 to
92, 111 and 112. [0284] 67. A protein comprising an ankyrin repeat
domain, wherein said ankyrin repeat domain comprises an N-terminal
capping module, one or more internal ankyrin repeats and a
C-terminal capping module, [0285] wherein said N-terminal capping
module has an amino acid sequence that has at least 95% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16,
[0286] wherein the amino acid residue of said N-terminal capping
module corresponding to position 15 in SEQ ID NO: 3 is selected
from the group consisting of L, M, I, T, A, V and S, [0287] wherein
said one or more internal ankyrin repeats each have an amino acid
sequence that has at least 95% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 77 to
82, and [0288] wherein said C-terminal capping module has an amino
acid sequence that has at least 95% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID NOs: 83
to 92, 111 and 112. [0289] 68. A protein comprising an ankyrin
repeat domain, wherein said ankyrin repeat domain comprises an
N-terminal capping module, one or more internal ankyrin repeats and
a C-terminal capping module, [0290] wherein said N-terminal capping
module has an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 to 16, [0291] wherein the amino acid
residue of said N-terminal capping module corresponding to position
15 in SEQ ID NO: 3 is selected from the group consisting of L, M,
I, T, A, V and S, [0292] wherein said one or more internal ankyrin
repeats each have an amino acid sequence selected from the group
consisting of SEQ ID NOs: 77 to 82, and [0293] wherein said
C-terminal capping module has an amino acid sequence selected from
the group consisting of SEQ ID NOs: 83 to 92, 111 and 112. [0294]
69. The protein according to any one of items 1 to 68, wherein said
ankyrin repeat domain has a higher melting temperature than a
reference ankyrin repeat domain having the same amino acid sequence
except for the amino acid residue of the N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3, which is selected
from the group consisting of E, D, G, H, K and N in the reference
ankyrin repeat domain. [0295] 70. The protein according to any one
of items 1 to 68, wherein said ankyrin repeat domain has a higher
melting temperature than a reference ankyrin repeat domain having
the same amino acid sequence except for the amino acid residue of
the N-terminal capping module corresponding to position 15 in SEQ
ID NO: 3, which is D in the reference ankyrin repeat domain. [0296]
71. The protein according to any one of items 1 to 68, wherein said
ankyrin repeat domain has a higher melting temperature than a
reference ankyrin repeat domain having the same amino acid sequence
except for the amino acid residue of the N-terminal capping module
corresponding to position 15 in SEQ ID NO: 3, which is E in the
reference ankyrin repeat domain. [0297] 72. The protein according
to any one of items 1 to 71, wherein said protein is a recombinant
protein. [0298] 73. The protein according to any one of items 1 to
72, wherein said ankyrin repeat domain specifically binds to a
target. [0299] 74. The protein according to item 73, wherein said
target is a virus. [0300] 75. The protein according to any one of
items 1 to 74, wherein said protein comprises one or more further
moieties in addition to said ankyrin repeat domain. [0301] 76. The
protein according to item 75, wherein said protein comprises one or
more additional ankyrin repeat domains as further moieties. [0302]
77. The protein according to item 76, wherein all of said ankyrin
repeat domains comprise the same N-terminal capping module. [0303]
78. The protein according to item 76 or 77, wherein said protein
comprises one or more additional ankyrin repeat domains as further
moieties that are connected by a proline-threonine linker. [0304]
79. The protein according to item 78, wherein the proline-threonine
linker has a length of 2 to 24 amino acid residues. [0305] 80. The
protein according to any one of items 1 to 79, wherein said protein
comprises at least five ankyrin repeat domains. [0306] 81. The
protein according to any one of items 1 to 75, wherein said protein
only comprises a single ankyrin repeat domain. [0307] 82. A protein
library comprising more than one protein according to any one of
items 1 to 81. [0308] 83. The protein library according to item 82,
wherein said library comprises at least 10.sup.7 proteins according
to any one of items 1 to 81, wherein said proteins all differ in
the amino acid sequence of their ankyrin repeat domain. [0309] 84.
The protein library according to item 82 or 83, wherein the one or
more proteins according to any one of items 1 to 81 share at least
70% sequence identity in their ankyrin repeat domain. [0310] 85. A
method for selecting a protein having a predetermined property
comprising the following steps: [0311] a. providing the protein
library of any one of items 82 to 84; [0312] b. selecting a protein
having the predetermined property from the protein library. [0313]
86. A method for selecting a protein having binding specificity to
a target comprising the following steps: [0314] a. providing the
protein library of any one of items 82 to 84; [0315] b. selecting a
protein having binding specificity to the target from the protein
library. [0316] 87. The method according to item 85 or 86,
comprising: [0317] c. Further modifying the amino acid sequence of
said selected protein. [0318] 88. A nucleic acid molecule
comprising a sequence encoding the protein according to any one of
items 1 to 81. [0319] 89. The nucleic acid molecule according to
item 88, wherein the nucleic acid molecule is a vector or a
chromosome. [0320] 90. A nucleic acid library comprising more than
one nucleic acid molecule according to item 88. [0321] 91. A cell
comprising the nucleic acid molecule according to item 88 or 89.
[0322] 92. A method of preparing a protein comprising the following
steps: [0323] a. culturing a cell comprising a nucleic acid
encoding a protein according to any one of items 1 to 81 under
conditions allowing expression of said protein; and [0324] b.
purifying the expressed protein. [0325] 93. A method of preparing a
protein comprising an ankyrin repeat domain with an improved
thermostability comprising the following steps: [0326] a. selecting
a protein comprising an ankyrin repeat domain, [0327] b.
determining the amino acid sequence of the N-terminal capping
module of said ankyrin repeat domain; and [0328] c. substituting
the amino acid residue of said N-terminal capping module
corresponding to position 15 of SEQ ID NO: 3 by an amino acid
residue selected from the group consisting of I, T, A, V, L and M.
[0329] 94. A method of preparing a protein comprising an ankyrin
repeat domain with an improved thermostability comprising the
following steps: [0330] a. selecting a protein comprising an
ankyrin repeat domain having an amino acid residue which is none of
I, T, A, V, L and M at the position of the N-terminal capping
module corresponding to position 15 of SEQ ID NO: 3; and [0331] b.
substituting the amino acid residue in said position by an amino
acid residue selected from the group consisting of I, T, A, V, L
and M. [0332] 95. The method of item 93 or 94 comprising the
further step of substituting the amino acid residue of said
N-terminal capping module corresponding to position 22 by an amino
acid selected from the group consisting of L, I and V. [0333] 96. A
pharmaceutical composition comprising a protein according to any
one of items 1 to 81 and a pharmaceutically acceptable carrier.
[0334] 97. A protein according to any one of items 1 to 81 for use
in a method of treatment. [0335] 98. Use of a protein according to
any one of items 1 to 81 for the manufacture of a medicament.
[0336] 99. A method of treatment comprising administering the
protein according to any one of items 1 to 81 to a subject.
EXAMPLES
Example 1: Effect of Mutations in the N-Terminal Capping Module on
the Thermostability of the Ankyrin Repeat Domain
[0337] Based on crystal structure data, each position of the
N-terminal capping module of an ankyrin repeat domain was analyzed
and predictions were made about the most suitable amino acid
residues for each position. In light of the inherent difficulty to
correctly predict the role of the mutations in the N-terminal
capping module, various mutations were tested by in vitro
experimentation.
[0338] Materials and Methods
[0339] Protein Sequences
[0340] Amongst others, His-tagged ankyrin repeat domains P #93 to P
#109 corresponding to SEQ ID NOs: 93 to 109, respectively, were
tested.
[0341] The DNA sequence encoding each ankyrin repeat domain was
chemically synthesized and cloned into pQIq (Simon M. et al.,
Bioconjug Chem., 23(2), 279-86, 2012) expression vectors by
standard techniques.
[0342] Protein Expression
[0343] The ankyrin repeat domains were expressed in E. coli BL21 or
XL1-Blue cells and purified using their His-tag using standard
protocols known to the person skilled in the art. Briefly, 25 ml of
stationary overnight cultures (LB, 1% glucose, 100 mg/I of
ampicillin; 37.degree. C.) were used to inoculate 1 l cultures
(same medium). At an absorbance of about 1 at 600 nm, the cultures
were induced with 0.5 mM IPTG and incubated at 37.degree. C. for 4
h. The cultures were centrifuged and the resulting pellets were
resuspended in 40 ml of TBS500 (50 mM Tris-HCl, 500 mM NaCl, pH 8)
and sonicated. The lysate was recentrifuged, and glycerol (10%
(v/v) final concentration) and imidazole (20 mM final
concentration) were added to the resulting supernatant. The ankyrin
repeat domains were purified over a Ni-nitrilotriacetic acid column
(2.5 ml column volume) according to the manufacturer's instructions
(QIAgen, Germany). Up to 200 mg of highly soluble ankyrin repeat
domains were purified from one liter of E. coli culture with a
purity >95% as estimated from SDS-15% PAGE. Such purified
ankyrin repeat domains were used for further characterizations.
[0344] CD Measurement
[0345] The CD signal of the ankyrin repeat domains was recorded at
222 nm in a Jasco J-810 instrument (Jasco, Japan) while slowly
heating the ankyrin repeat domains at a concentration of 0.01 mM in
PBS pH 7.4 from 20.degree. C. to 95.degree. C. using a temperature
ramp of 1.degree. C. per min. This is an effective means to follow
the denaturation of ankyrin repeat domains as they mainly consist
of alpha helices that show a strong change in their CD signal at
222 nm upon unfolding. The midpoint of the observed transition of
such a measured CD signal trace for an ankyrin repeat domain
corresponds to its Tm value. Tm values were derived as described in
V. Consalvi et al. (Protein Eng Des Sel. 13, 501-507, 2000).
[0346] Results and Discussion
[0347] The melting curves for the above-mentioned ankyrin repeat
domains were determined. Based on the measured melting curves, the
Tm values of the various constructs were determined as described
above.
[0348] The ankyrin repeat domain P #95 corresponding to SEQ ID NO:
95 was used as a first exemplary reference ankyrin repeat domain. P
#95 comprises a D at position 27 (which corresponds to position 15
of its N-terminal capping module). P #96 to P #107 corresponding to
SEQ ID NOs: 96 to 107, respectively, only differ in the amino acid
residue at this position 15 of their N-terminal capping module from
P #95. FIG. 1 shows, as an example, the melting curves of P #95 and
P #101. Thus, changing a single amino acid at position 15 (from D
to V) resulted in a strong increase of the Tm value of the ankyrin
repeat protein of about 10.degree. C. (the Tm values for P #95 and
P #101 were estimated to be 74.5.degree. C. and 85.1.degree. C.,
respectively). Table 2 shows the Tm values and the corresponding
amino acids at position 15 of the respective N-terminal capping
modules of P #95 to P #107.
TABLE-US-00002 TABLE 2 Protein SEQ ID NO: Position 15 Tm value
[.degree. C.] P#95 95 D 74.5 P#96 96 L 84.6 P#97 97 M 83.8 P#98 98
I 84.8 P#99 99 T 82.3 P#100 100 A 82.4 P#101 101 V 85.1 P#102 102 S
79.3 P#103 103 N 75.2 P#104 104 Q 77.4 P#105 105 K 77.9 P#106 106 R
78.3 P#107 107 E 79.2
[0349] Only some of the amino acid residues in position 15, in
particular I, T, A, V, L and M resulted in a profound increase of
the Tm value for the ankyrin repeat protein, e.g. when compared to
D in this position 15.
[0350] A further reference ankyrin repeat domain (P #93) that was
tested corresponds to SEQ ID NO: 93. P #93 comprises a D at
position 27 (which corresponds to position 15 of its N-terminal
capping module). The ankyrin repeat domain P #94 corresponding to
SEQ ID NO: 94 is identical to P #93, with the exception that it
contains the amino acid L at this position 15. FIG. 2 shows the
melting curves of P #93 and P #94. Thus, changing a single amino
acid at position 15 (from D to L) resulted in a strong increase of
the Tm value of the ankyrin repeat protein of about 13.degree. C.
(the Tm values for P #93 and P #94 were estimated to be
62.1.degree. C. and 75.2.degree. C., respectively).
[0351] Yet another reference ankyrin repeat domain (P #108) that
was tested corresponds to SEQ ID NO: 108. P #108 comprises a D at
position 27 (which corresponds to position 15 of its N-terminal
capping module). The ankyrin repeat domain P #109 corresponding to
SEQ ID NO: 109 is identical to P #108, with the exception that it
contains the amino acid L at this position 15. FIG. 3 shows the
melting curves of P #108 and P #109. Thus, changing a single amino
acid at position 15 (from D to L) resulted in a strong increase of
the Tm value of the ankyrin repeat protein of about 14.degree. C.
(the Tm values for P #108 and P #109 were estimated to be
68.6.degree. C. and 82.8.degree. C., respectively).
[0352] Even though all three reference ankyrin repeat domains used
(i.e. P #93, P #95 and P #109) significantly differ in their amino
acid sequences of their N-terminal capping module (up to 9 amino
acid differences outside position 15) a single mutation at position
15 can result in a surprisingly strong Tm increase of over
10.degree. C. of the respective ankyrin repeat domain.
[0353] Overall, these data show that mutations at position 15 of a
N-terminal capping module, in particular I, T, A, V, L and M, are
beneficial for the thermostability of ankyrin repeat domains, incl.
designed ankyrin repeat proteins or DARPins.
[0354] The stabilizing effect of such mutations in position 15 of
the N-terminal capping module were further confirmed using
different ankyrin repeat domains.
Example 2: Effect of Mutations in the N-Terminal Capping Module on
the Thermostability of the Ankyrin Repeat Domain
[0355] To confirm the stabilizing effect of the mutations in the
N-terminal capping module, the mutations were tested in the context
of further ankyrin repeat proteins.
[0356] Materials and Methods
[0357] Protein Sequences and Expression
[0358] His-tagged ankyrin repeat domains P #113 to P #125
corresponding to SEQ ID NOs: 113 to 125, respectively, were
tested.
[0359] Cloning and expression of the ankyrin repeat domains was
done as described in Example 1.
[0360] CD Measurement
[0361] The CD measurements of the ankyrin repeat domains P #122 to
P #125 were done as described in Example 1.
[0362] The CD signal of the ankyrin repeat domains P #113 to P #121
was recorded at 222 nm in a Chirascan V100 instrument (Applied
Photophysics) while slowly heating the ankyrin repeat domains at a
concentration of 0.01 mM in PBS, 2M GdmCl, pH 7.4 from 25.degree.
C. to 100.degree. C. using a temperature ramp of 1.degree. C. per
min, collecting data periodically at 0.5.degree. C. intervals. Tm
values were derived from the CD signal as described in Example
1.
[0363] Results and Discussion
[0364] The mutations at position 15 were transferred to three
ankyrin repeat domains with different binding specificities and
largely diverging sequences. In particular, the ankyrin repeat
domain P #113 corresponding to SEQ ID NO: 113 specifically binds to
human serum albumin (HSA), the ankyrin repeat domain P #116
corresponding to SEQ ID NO: 116 specifically binds to human
vascular endothelial growth factor (VEGF) and the ankyrin repeat
domain P #119 corresponding to SEQ ID NO: 119 specifically binds to
human epidermal growth factor receptor 2 (HER2). In each of these
ankyrin repeat domains, the D at position 27 of the respective
sequences (which corresponds to position 15 in the N-terminal
capping module) was replaced by V and L, respectively.
[0365] The Tm values of these ankyrin repeat domains are summarized
in Table 3:
TABLE-US-00003 TABLE 3 Protein SEQ ID NO: Target Position 15 Tm
value [.degree. C.] P#113 113 HSA D 76.0 P#114 114 HSA L 84.1 P#115
115 HSA V 81.9 P#116 116 VEGF D unfolded at RT of about 22.degree.
C. P#117 117 VEGF L 45.1 P#118 118 VEGF V 42 P#119 119 HER2 D 39.6
P#120 120 HER2 L 55.7 P#121 121 HER2 V 52.6
[0366] As reflected by the above table, replacing the amino acid
residue at position 15 of the N-terminal capping module by either
of V or L increases the thermostability of the ankyrin repeat
domain in all tested backgrounds significantly.
[0367] The particular suitability of the mutations at position 15
of the N-terminal capping module was further confirmed in different
ankyrin repeat domain backgrounds, including ankyrin repeat domain
P #122 corresponding to SEQ ID NO: 122 (Tm of 83.7.degree. C.),
ankyrin repeat domain P #123 corresponding to SEQ ID NO: 123 (Tm of
83.9.degree. C.), ankyrin repeat domain P #124 corresponding to SEQ
ID NO: 124 (Tm of 81.7.degree. C.) and ankyrin repeat domain P #125
corresponding to SEQ ID NO: 125 (Tm of 84.2.degree. C.).
Sequence CWU 1
1
125130PRTArtificial SequenceSynthetic Construct 1Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp1 5 10 15Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
30230PRTArtificial SequenceSynthetic Construct 2Asp Leu Gly Lys Lys
Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp1 5 10 15Glu Val Arg Ile
Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30330PRTArtificial
SequenceSynthetic Construct 3Asp Leu Gly Lys Lys Leu Leu Glu Ala
Ala Arg Ala Gly Gln Asp Asp1 5 10 15Glu Val Arg Ile Leu Met Ala Asn
Gly Ala Asp Val Asn Ala 20 25 30430PRTArtificial SequenceSynthetic
Construct 4Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln
Asp Asp1 5 10 15Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn
Ala 20 25 30530PRTArtificial SequenceSynthetic Construct 5Asp Leu
Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp1 5 10 15Glu
Val Arg Ile Leu Ile Ala Asn Gly Ala Pro Phe Thr Thr 20 25
30630PRTArtificial SequenceSynthetic Construct 6Asp Leu Gly Lys Lys
Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp1 5 10 15Glu Val Arg Glu
Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25 30730PRTArtificial
SequenceSynthetic ConstructVARIANT15Xaa can be any naturally
occuring amino acid 7Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg
Ala Gly Gln Xaa Asp1 5 10 15Glu Val Arg Ile Leu Met Ala Asn Gly Ala
Asp Val Asn Ala 20 25 30830PRTArtificial SequenceSynthetic
ConstructVARIANT15Xaa can be any naturally occuring amino acid 8Asp
Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Xaa Asp1 5 10
15Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
30930PRTArtificial SequenceSynthetic ConstructVARIANT15Xaa can be
any naturally occuring amino acid 9Asp Leu Asp Lys Lys Leu Leu Glu
Ala Ala Arg Ala Gly Gln Xaa Asp1 5 10 15Glu Val Arg Glu Leu Thr Lys
Ala Gly Ala Asp Val Asn Ala 20 25 301030PRTArtificial
SequenceSynthetic ConstructVARIANT15Xaa can be any naturally
occuring amino acid 10Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg
Ala Gly Gln Xaa Asp1 5 10 15Glu Val Arg Glu Leu Ile Lys Ala Gly Ala
Asp Val Asn Ala 20 25 301130PRTArtificial SequenceSynthetic
ConstructVARIANT15Xaa can be any naturally occuring amino acid
11Asp Leu Asp Lys Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Xaa Asp1
5 10 15Ala Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20
25 301230PRTArtificial SequenceSynthetic ConstructVARIANT15Xaa can
be any naturally occuring amino acid 12Asp Leu Ala Lys Lys Leu Leu
Glu Ala Ala Ala Ala Gly Gln Xaa Asp1 5 10 15Ala Val Arg Glu Leu Val
Lys Ala Gly Ala Asp Val Asn Ala 20 25 301330PRTArtificial
SequenceSynthetic ConstructVARIANT15Xaa can be any naturally
occuring amino acid 13Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Ala
Ala Gly Gln Xaa Asp1 5 10 15Ala Val Arg Glu Leu Ile Lys Ala Gly Ala
Asp Val Asn Ala 20 25 301430PRTArtificial SequenceSynthetic
ConstructVARIANT15Xaa can be any naturally occuring amino acid
14Asp Leu Ala Lys Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Xaa Asp1
5 10 15Ala Val Arg Glu Leu Ala Lys Ala Gly Ala Asp Val Asn Ala 20
25 301530PRTArtificial SequenceSynthetic ConstructVARIANT15Xaa can
be any naturally occuring amino acid 15Asp Leu Ala Lys Lys Leu Leu
Ala Ala Ala Ala Ala Gly Gln Xaa Glu1 5 10 15Ala Val Glu Gln Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala 20 25 301630PRTArtificial
SequenceSynthetic ConstructVARIANT15Xaa can be any naturally
occuring amino acid 16Asp Leu Gly Lys Lys Leu Leu Ala Ala Ala Ala
Ala Gly Gln Xaa Glu1 5 10 15Ala Val Glu Gln Leu Ile Lys Ala Gly Ala
Asp Val Asn Ala 20 25 301730PRTArtificial SequenceSynthetic
Construct 17Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln
Ile Asp1 5 10 15Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn
Ala 20 25 301830PRTArtificial SequenceSynthetic Construct 18Asp Leu
Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ile Asp1 5 10 15Glu
Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
301930PRTArtificial SequenceSynthetic Construct 19Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ile Asp1 5 10 15Glu Val Arg
Glu Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25
302030PRTArtificial SequenceSynthetic Construct 20Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ile Asp1 5 10 15Glu Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
302130PRTArtificial SequenceSynthetic Construct 21Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ile Asp1 5 10 15Ala Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
302230PRTArtificial SequenceSynthetic Construct 22Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ile Asp1 5 10 15Ala Val Arg
Glu Leu Val Lys Ala Gly Ala Asp Val Asn Ala 20 25
302330PRTArtificial SequenceSynthetic Construct 23Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ile Asp1 5 10 15Ala Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
302430PRTArtificial SequenceSynthetic Construct 24Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ile Asp1 5 10 15Ala Val Arg
Glu Leu Ala Lys Ala Gly Ala Asp Val Asn Ala 20 25
302530PRTArtificial SequenceSynthetic Construct 25Asp Leu Ala Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Ile Glu1 5 10 15Ala Val Glu
Gln Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
302630PRTArtificial SequenceSynthetic Construct 26Asp Leu Gly Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Ile Glu1 5 10 15Ala Val Glu
Gln Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
302730PRTArtificial SequenceSynthetic Construct 27Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Thr Asp1 5 10 15Glu Val Arg
Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala 20 25
302830PRTArtificial SequenceSynthetic Construct 28Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Thr Asp1 5 10 15Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
302930PRTArtificial SequenceSynthetic Construct 29Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Thr Asp1 5 10 15Glu Val Arg
Glu Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25
303030PRTArtificial SequenceSynthetic Construct 30Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Thr Asp1 5 10 15Glu Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
303130PRTArtificial SequenceSynthetic Construct 31Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Thr Asp1 5 10 15Ala Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
303230PRTArtificial SequenceSynthetic Construct 32Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Thr Asp1 5 10 15Ala Val Arg
Glu Leu Val Lys Ala Gly Ala Asp Val Asn Ala 20 25
303330PRTArtificial SequenceSynthetic Construct 33Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Thr Asp1 5 10 15Ala Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
303430PRTArtificial SequenceSynthetic Construct 34Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Thr Asp1 5 10 15Ala Val Arg
Glu Leu Ala Lys Ala Gly Ala Asp Val Asn Ala 20 25
303530PRTArtificial SequenceSynthetic Construct 35Asp Leu Ala Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Thr Glu1 5 10 15Ala Val Glu
Gln Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
303630PRTArtificial SequenceSynthetic Construct 36Asp Leu Gly Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Thr Glu1 5 10 15Ala Val Glu
Gln Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
303730PRTArtificial SequenceSynthetic Construct 37Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ala Asp1 5 10 15Glu Val Arg
Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala 20 25
303830PRTArtificial SequenceSynthetic Construct 38Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ala Asp1 5 10 15Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
303930PRTArtificial SequenceSynthetic Construct 39Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ala Asp1 5 10 15Glu Val Arg
Glu Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25
304030PRTArtificial SequenceSynthetic Construct 40Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ala Asp1 5 10 15Glu Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
304130PRTArtificial SequenceSynthetic Construct 41Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ala Asp1 5 10 15Ala Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
304230PRTArtificial SequenceSynthetic Construct 42Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ala Asp1 5 10 15Ala Val Arg
Glu Leu Val Lys Ala Gly Ala Asp Val Asn Ala 20 25
304330PRTArtificial SequenceSynthetic Construct 43Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ala Asp1 5 10 15Ala Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
304430PRTArtificial SequenceSynthetic Construct 44Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Ala Asp1 5 10 15Ala Val Arg
Glu Leu Ala Lys Ala Gly Ala Asp Val Asn Ala 20 25
304530PRTArtificial SequenceSynthetic Construct 45Asp Leu Ala Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Ala Glu1 5 10 15Ala Val Glu
Gln Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
304630PRTArtificial SequenceSynthetic Construct 46Asp Leu Gly Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Ala Glu1 5 10 15Ala Val Glu
Gln Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
304730PRTArtificial SequenceSynthetic Construct 47Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp1 5 10 15Glu Val Arg
Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala 20 25
304830PRTArtificial SequenceSynthetic Construct 48Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp1 5 10 15Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
304930PRTArtificial SequenceSynthetic Construct 49Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp1 5 10 15Glu Val Arg
Glu Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25
305030PRTArtificial SequenceSynthetic Construct 50Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp1 5 10 15Glu Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
305130PRTArtificial SequenceSynthetic Construct 51Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Val Asp1 5 10 15Ala Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
305230PRTArtificial SequenceSynthetic Construct 52Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Val Asp1 5 10 15Ala Val Arg
Glu Leu Val Lys Ala Gly Ala Asp Val Asn Ala 20 25
305330PRTArtificial SequenceSynthetic Construct 53Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Val Asp1 5 10 15Ala Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
305430PRTArtificial SequenceSynthetic Construct 54Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Val Asp1 5 10 15Ala Val Arg
Glu Leu Ala Lys Ala Gly Ala Asp Val Asn Ala 20 25
305530PRTArtificial SequenceSynthetic Construct 55Asp Leu Ala Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Val Glu1 5 10 15Ala Val Glu
Gln Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
305630PRTArtificial SequenceSynthetic Construct 56Asp Leu Gly Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Val Glu1 5 10 15Ala Val Glu
Gln Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
305730PRTArtificial SequenceSynthetic Construct 57Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp1 5 10 15Glu Val Arg
Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala 20 25
305830PRTArtificial SequenceSynthetic Construct 58Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp1 5 10 15Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
305930PRTArtificial SequenceSynthetic Construct 59Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp1 5 10 15Glu Val Arg
Glu Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25
306030PRTArtificial SequenceSynthetic Construct 60Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp1 5 10 15Glu Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
306130PRTArtificial SequenceSynthetic Construct 61Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Leu Asp1 5 10 15Ala Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
306230PRTArtificial SequenceSynthetic Construct 62Asp Leu Ala Lys
Lys Leu
Leu Glu Ala Ala Ala Ala Gly Gln Leu Asp1 5 10 15Ala Val Arg Glu Leu
Val Lys Ala Gly Ala Asp Val Asn Ala 20 25 306330PRTArtificial
SequenceSynthetic Construct 63Asp Leu Gly Lys Lys Leu Leu Glu Ala
Ala Ala Ala Gly Gln Leu Asp1 5 10 15Ala Val Arg Glu Leu Ile Lys Ala
Gly Ala Asp Val Asn Ala 20 25 306430PRTArtificial SequenceSynthetic
Construct 64Asp Leu Ala Lys Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln
Leu Asp1 5 10 15Ala Val Arg Glu Leu Ala Lys Ala Gly Ala Asp Val Asn
Ala 20 25 306530PRTArtificial SequenceSynthetic Construct 65Asp Leu
Ala Lys Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Leu Glu1 5 10 15Ala
Val Glu Gln Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
306630PRTArtificial SequenceSynthetic Construct 66Asp Leu Gly Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Leu Glu1 5 10 15Ala Val Glu
Gln Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
306730PRTArtificial SequenceSynthetic Construct 67Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Met Asp1 5 10 15Glu Val Arg
Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala 20 25
306830PRTArtificial SequenceSynthetic Construct 68Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Met Asp1 5 10 15Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
306930PRTArtificial SequenceSynthetic Construct 69Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Met Asp1 5 10 15Glu Val Arg
Glu Leu Thr Lys Ala Gly Ala Asp Val Asn Ala 20 25
307030PRTArtificial SequenceSynthetic Construct 70Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Met Asp1 5 10 15Glu Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
307130PRTArtificial SequenceSynthetic Construct 71Asp Leu Asp Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Met Asp1 5 10 15Ala Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
307230PRTArtificial SequenceSynthetic Construct 72Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Met Asp1 5 10 15Ala Val Arg
Glu Leu Val Lys Ala Gly Ala Asp Val Asn Ala 20 25
307330PRTArtificial SequenceSynthetic Construct 73Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Met Asp1 5 10 15Ala Val Arg
Glu Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
307430PRTArtificial SequenceSynthetic Construct 74Asp Leu Ala Lys
Lys Leu Leu Glu Ala Ala Ala Ala Gly Gln Met Asp1 5 10 15Ala Val Arg
Glu Leu Ala Lys Ala Gly Ala Asp Val Asn Ala 20 25
307530PRTArtificial SequenceSynthetic Construct 75Asp Leu Ala Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Met Glu1 5 10 15Ala Val Glu
Gln Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
307630PRTArtificial SequenceSynthetic Construct 76Asp Leu Gly Lys
Lys Leu Leu Ala Ala Ala Ala Ala Gly Gln Met Glu1 5 10 15Ala Val Glu
Gln Leu Ile Lys Ala Gly Ala Asp Val Asn Ala 20 25
307733PRTArtificial SequenceSynthetic ConstructVARIANT1Xaa can be
any naturally occuring amino acidVARIANT3Xaa can be any naturally
occuring amino acidVARIANT4Xaa can be any naturally occuring amino
acidVARIANT6Xaa can be any naturally occuring amino
acidVARIANT13Xaa can be any naturally occuring amino
acidVARIANT14Xaa can be any naturally occuring amino
acidVARIANT15Xaa can be any naturally occuring amino
acidVARIANT17Xaa can be any naturally occuring amino
acidVARIANT18Xaa can be any naturally occuring amino
acidVARIANT19Xaa can be any naturally occuring amino
acidVARIANT22Xaa can be any naturally occuring amino
acidVARIANT26Xaa can be any naturally occuring amino
acidVARIANT27Xaa can be any naturally occuring amino acid 77Xaa Asp
Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Xaa Xaa Xaa Gly1 5 10 15Xaa
Xaa Xaa Val Val Xaa Leu Leu Leu Xaa Xaa Gly Ala Asp Val Asn 20 25
30Ala7833PRTArtificial SequenceSynthetic ConstructVARIANT1Xaa can
be any naturally occuring amino acidVARIANT3Xaa can be any
naturally occuring amino acidVARIANT4Xaa can be any naturally
occuring amino acidVARIANT6Xaa can be any naturally occuring amino
acidVARIANT14Xaa can be any naturally occuring amino
acidVARIANT15Xaa can be any naturally occuring amino
acidVARIANT27Xaa can be any naturally occuring amino acid 78Xaa Asp
Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly1 5 10 15His
Leu Glu Ile Val Glu Val Leu Leu Lys Xaa Gly Ala Asp Val Asn 20 25
30Ala7933PRTArtificial SequenceSynthetic ConstructVARIANT2Xaa can
be any naturally occuring amino acidVARIANT3Xaa can be any
naturally occuring amino acidVARIANT4Xaa can be any naturally
occuring amino acidVARIANT6Xaa can be any naturally occuring amino
acidVARIANT11Xaa can be any naturally occuring amino
acidVARIANT14Xaa can be any naturally occuring amino
acidVARIANT15Xaa can be any naturally occuring amino
acidVARIANT27Xaa can be any naturally occuring amino acid 79Ser Xaa
Xaa Xaa Gly Xaa Thr Pro Leu His Xaa Ala Ala Xaa Xaa Gly1 5 10 15His
Leu Glu Ile Val Asp Val Leu Leu Ala Xaa Gly Ala Asp Val Asn 20 25
30Ala8033PRTArtificial SequenceSynthetic ConstructVARIANT1Xaa can
be any naturally occuring amino acidVARIANT3Xaa can be any
naturally occuring amino acidVARIANT4Xaa can be any naturally
occuring amino acidVARIANT6Xaa can be any naturally occuring amino
acidVARIANT14Xaa can be any naturally occuring amino
acidVARIANT15Xaa can be any naturally occuring amino acid 80Xaa Asp
Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly1 5 10 15His
Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn 20 25
30Ala8133PRTArtificial SequenceSynthetic ConstructVARIANT3Xaa can
be any naturally occuring amino acidVARIANT4Xaa can be any
naturally occuring amino acidVARIANT6Xaa can be any naturally
occuring amino acidVARIANT14Xaa can be any naturally occuring amino
acidVARIANT15Xaa can be any naturally occuring amino acid 81Lys Asp
Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly1 5 10 15His
Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 20 25
30Ala8233PRTArtificial SequenceSynthetic ConstructVARIANT1Xaa can
be any naturally occuring amino acidVARIANT3Xaa can be any
naturally occuring amino acidVARIANT4Xaa can be any naturally
occuring amino acidVARIANT6Xaa can be any naturally occuring amino
acidVARIANT11Xaa can be any naturally occuring amino
acidVARIANT14Xaa can be any naturally occuring amino
acidVARIANT15Xaa can be any naturally occuring amino
acidVARIANT27Xaa can be any naturally occuring amino acid 82Xaa Asp
Xaa Xaa Gly Xaa Thr Pro Leu His Xaa Ala Ala Xaa Xaa Gly1 5 10 15His
Leu Glu Val Val Lys Leu Leu Leu Glu Xaa Gly Ala Asp Val Asn 20 25
30Ala8328PRTArtificial SequenceSynthetic Construct 83Gln Asp Lys
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly1 5 10 15Asn Glu
Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn 20 258428PRTArtificial
SequenceSynthetic Construct 84Gln Asp Lys Phe Gly Lys Thr Ala Phe
Asp Ile Ser Ile Asp Asn Gly1 5 10 15Asn Glu Asp Leu Ala Glu Ile Leu
Gln Lys Ala Ala 20 258528PRTArtificial SequenceSynthetic Construct
85Gln Asp Lys Phe Gly Lys Thr Pro Phe Asp Leu Ala Ile Asp Asn Gly1
5 10 15Asn Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 20
258628PRTArtificial SequenceSynthetic Construct 86Gln Asp Lys Phe
Gly Lys Thr Pro Phe Asp Leu Ala Ile Arg Glu Gly1 5 10 15His Glu Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala 20 258728PRTArtificial
SequenceSynthetic Construct 87Gln Asp Lys Ser Gly Lys Thr Pro Ala
Asp Leu Ala Ala Asp Ala Gly1 5 10 15His Glu Asp Ile Ala Glu Val Leu
Gln Lys Ala Ala 20 258828PRTArtificial SequenceSynthetic Construct
88Gln Asp Ser Ala Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly1
5 10 15His Glu Glu Ile Ala Glu Val Leu Gln Lys Ala Ala 20
258928PRTArtificial SequenceSynthetic Construct 89Gln Asp Lys Ala
Gly Leu Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly1 5 10 15His Glu Glu
Ile Ala Glu Val Leu Gln Lys Ala Ala 20 259028PRTArtificial
SequenceSynthetic Construct 90Gln Asp Ser Leu Gly Lys Thr Pro Ala
Asp Leu Ala Ala Leu Ala Gly1 5 10 15His Glu Glu Ile Ala Glu Val Leu
Gln Lys Ala Ala 20 259128PRTArtificial SequenceSynthetic Construct
91Gln Asp Lys Ala Gly Gln Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly1
5 10 15His Glu Glu Ile Ala Glu Val Leu Gln Lys Ala Ala 20
259228PRTArtificial SequenceSynthetic ConstructVARIANT3Xaa can be
any naturally occuring amino acidVARIANT4Xaa can be any naturally
occuring amino acidVARIANT6Xaa can be any naturally occuring amino
acidVARIANT14Xaa can be any naturally occuring amino acid 92Gln Asp
Xaa Xaa Gly Xaa Thr Pro Ala Asp Leu Ala Ala Xaa Ala Gly1 5 10 15His
Glu Glu Ile Ala Glu Val Leu Gln Lys Ala Ala 20 2593103PRTArtificial
SequenceSynthetic Construct 93Met Arg Gly Ser His His His His His
His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln Asp Asp Glu Val Arg Ile 20 25 30Leu Met Ala Asn Gly Ala Asp
Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala
Ala Arg Glu Gly His Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala
Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile
Leu Gln Lys Leu Asn 10094103PRTArtificial SequenceSynthetic
Construct 94Met Arg Gly Ser His His His His His His Gly Ser Asp Leu
Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp Glu
Val Arg Ile 20 25 30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp
Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His
Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile
Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
10095103PRTArtificial SequenceSynthetic Construct 95Met Arg Gly Ser
His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu
Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu 20 25 30Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40 45Thr
Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu 50 55
60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65
70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu
Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn 10096103PRTArtificial
SequenceSynthetic Construct 96Met Arg Gly Ser His His His His His
His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln Leu Asp Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala
Ala Arg Glu Gly His Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala
Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile
Leu Gln Lys Leu Asn 10097103PRTArtificial SequenceSynthetic
Construct 97Met Arg Gly Ser His His His His His His Gly Ser Asp Leu
Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Met Asp Glu
Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His
Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile
Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
10098103PRTArtificial SequenceSynthetic Construct 98Met Arg Gly Ser
His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu
Glu Ala Ala Arg Ala Gly Gln Ile Asp Glu Val Arg Glu 20 25 30Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40 45Thr
Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu 50 55
60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65
70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu
Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn 10099103PRTArtificial
SequenceSynthetic Construct 99Met Arg Gly Ser His His His His His
His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln Thr Asp Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala
Ala Arg Glu Gly His Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala
Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile
Leu Gln Lys Leu Asn 100100103PRTArtificial SequenceSynthetic
Construct 100Met Arg Gly Ser His His His His His His Gly Ser Asp
Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Ala Asp
Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
Asp Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly
His Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser
Ile Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu
Asn 100101103PRTArtificial SequenceSynthetic Construct 101Met Arg
Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys
Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp Glu Val Arg Glu 20 25
30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr
35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val
Glu 50 55
60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65
70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu
Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn 100102103PRTArtificial
SequenceSynthetic Construct 102Met Arg Gly Ser His His His His His
His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln Ser Asp Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala
Ala Arg Glu Gly His Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala
Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile
Leu Gln Lys Leu Asn 100103103PRTArtificial SequenceSynthetic
Construct 103Met Arg Gly Ser His His His His His His Gly Ser Asp
Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asn Asp
Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
Asp Lys Asp Gly Tyr 35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly
His Leu Glu Ile Val Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Lys Phe Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser
Ile Asp Asn Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu
Asn 100104103PRTArtificial SequenceSynthetic Construct 104Met Arg
Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys
Leu Leu Glu Ala Ala Arg Ala Gly Gln Gln Asp Glu Val Arg Glu 20 25
30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr
35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val
Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys
Phe Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn
Glu Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100105103PRTArtificial SequenceSynthetic Construct 105Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Lys Asp Glu Val Arg Glu 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100106103PRTArtificial SequenceSynthetic Construct 106Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Arg Asp Glu Val Arg Glu 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100107103PRTArtificial SequenceSynthetic Construct 107Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Glu Asp Glu Val Arg Glu 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100108103PRTArtificial SequenceSynthetic Construct 108Met Arg Gly
Ser His His His His His His Asp Ala Asp Leu Ala Lys1 5 10 15Lys Leu
Leu Ala Ala Ala Ala Ala Gly Gln Asp Glu Ala Val Glu Gln 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100109103PRTArtificial SequenceSynthetic Construct 109Met Arg Gly
Ser His His His His His His Asp Ala Asp Leu Ala Lys1 5 10 15Lys Leu
Leu Ala Ala Ala Ala Ala Gly Gln Leu Glu Ala Val Glu Gln 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
10011030PRTArtificial SequenceSynthetic Construct 110Asp Leu Gly
Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp1 5 10 15Glu Val
Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
3011128PRTArtificial SequenceSynthetic construct 111Gln Asp Lys Ser
Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly1 5 10 15His Gln Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala 20 2511228PRTArtificial
SequenceSynthetic Construct 112Gln Asp Lys Phe Gly Lys Thr Ala Glu
Asp Leu Ala Lys Asp Asn Gly1 5 10 15Asn Gln Asp Ile Ala Asp Leu Leu
Glu Lys Ala Leu 20 25113136PRTArtificial SequenceSynthetic
Construct 113Met Arg Gly Ser His His His His His His Gly Ser Asp
Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
Asp Tyr Phe Ser His 35 40 45Thr Pro Leu His Leu Ala Ala Arg Asn Gly
His Leu Lys Ile Val Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Phe Ala Gly65 70 75 80Lys Thr Pro Leu His Leu Ala
Ala Asn Glu Gly His Leu Glu Ile Val 85 90 95Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Ile Phe 100 105 110Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile 115 120 125Ala Glu
Val Leu Gln Lys Ala Ala 130 135114136PRTArtificial
SequenceSynthetic Construct 114Met Arg Gly Ser His His His His His
His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln Leu Asp Glu Val Arg Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys Asp Tyr Phe Ser His 35 40 45Thr Pro Leu His Leu Ala
Ala Arg Asn Gly His Leu Lys Ile Val Glu 50 55 60Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Phe Ala Gly65 70 75 80Lys Thr Pro
Leu His Leu Ala Ala Asn Glu Gly His Leu Glu Ile Val 85 90 95Glu Val
Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Phe 100 105
110Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp Ile
115 120 125Ala Glu Val Leu Gln Lys Ala Ala 130
135115136PRTArtificial SequenceSynthetic Construct 115Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Val Asp Glu Val Arg Glu 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser His 35 40
45Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala
Gly65 70 75 80Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His Leu
Glu Ile Val 85 90 95Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Gln Asp Ile Phe 100 105 110Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp
Ala Gly His Glu Asp Ile 115 120 125Ala Glu Val Leu Gln Lys Ala Ala
130 135116136PRTArtificial SequenceSynthetic Construct 116Met Arg
Gly Ser His His His His His His Gly Ser Asp Leu Asp Lys1 5 10 15Lys
Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile 20 25
30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Arg Asp Ser Thr Gly Trp
35 40 45Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro Glu Ile Val
Glu 50 55 60Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ala Asp Phe
Gln Gly65 70 75 80Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly His
Leu Glu Ile Val 85 90 95Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn
Ala Gln Asp Lys Phe 100 105 110Gly Lys Thr Ala Phe Asp Ile Ser Ile
Asp Asn Gly Asn Glu Asp Leu 115 120 125Ala Glu Ile Leu Gln Lys Ala
Ala 130 135117136PRTArtificial SequenceSynthetic Construct 117Met
Arg Gly Ser His His His His His His Gly Ser Asp Leu Asp Lys1 5 10
15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Ile
20 25 30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Arg Asp Ser Thr Gly
Trp 35 40 45Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro Glu Ile
Val Glu 50 55 60Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ala Asp
Phe Gln Gly65 70 75 80Trp Thr Pro Leu His Leu Ala Ala Ala Val Gly
His Leu Glu Ile Val 85 90 95Glu Val Leu Leu Lys Tyr Gly Ala Asp Val
Asn Ala Gln Asp Lys Phe 100 105 110Gly Lys Thr Ala Phe Asp Ile Ser
Ile Asp Asn Gly Asn Glu Asp Leu 115 120 125Ala Glu Ile Leu Gln Lys
Ala Ala 130 135118136PRTArtificial SequenceSynthetic Construct
118Met Arg Gly Ser His His His His His His Gly Ser Asp Leu Asp Lys1
5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp Glu Val Arg
Ile 20 25 30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Arg Asp Ser Thr
Gly Trp 35 40 45Thr Pro Leu His Leu Ala Ala Pro Trp Gly His Pro Glu
Ile Val Glu 50 55 60Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ala
Asp Phe Gln Gly65 70 75 80Trp Thr Pro Leu His Leu Ala Ala Ala Val
Gly His Leu Glu Ile Val 85 90 95Glu Val Leu Leu Lys Tyr Gly Ala Asp
Val Asn Ala Gln Asp Lys Phe 100 105 110Gly Lys Thr Ala Phe Asp Ile
Ser Ile Asp Asn Gly Asn Glu Asp Leu 115 120 125Ala Glu Ile Leu Gln
Lys Ala Ala 130 135119136PRTArtificial SequenceSynthetic Construct
119Met Arg Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys1
5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
Ile 20 25 30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr
Gly Leu 35 40 45Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu
Ile Val Glu 50 55 60Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Val
Asp Ala Ile Gly65 70 75 80Phe Thr Pro Leu His Leu Ala Ala Phe Ile
Gly His Leu Glu Ile Ala 85 90 95Glu Val Leu Leu Lys His Gly Ala Asp
Val Asn Ala Gln Asp Lys Phe 100 105 110Gly Lys Thr Ala Phe Asp Ile
Ser Ile Gly Asn Gly Asn Glu Asp Leu 115 120 125Ala Glu Ile Leu Gln
Lys Leu Asn 130 135120136PRTArtificial SequenceSynthetic Construct
120Met Arg Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys1
5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg
Ile 20 25 30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr
Gly Leu 35 40 45Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu
Ile Val Glu 50 55 60Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Val
Asp Ala Ile Gly65 70 75 80Phe Thr Pro Leu His Leu Ala Ala Phe Ile
Gly His Leu Glu Ile Ala 85 90 95Glu Val Leu Leu Lys His Gly Ala Asp
Val Asn Ala Gln Asp Lys Phe 100 105 110Gly Lys Thr Ala Phe Asp Ile
Ser Ile Gly Asn Gly Asn Glu Asp Leu 115 120 125Ala Glu Ile Leu Gln
Lys Leu Asn 130 135121136PRTArtificial SequenceSynthetic Construct
121Met Arg Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys1
5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Val Asp Glu Val Arg
Ile 20 25 30Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr
Gly Leu 35 40 45Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu
Ile Val Glu 50 55 60Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Val
Asp Ala Ile Gly65 70 75 80Phe Thr Pro Leu His Leu Ala Ala Phe Ile
Gly His Leu Glu Ile Ala 85 90 95Glu Val Leu Leu Lys His Gly Ala Asp
Val Asn Ala Gln Asp Lys Phe 100 105 110Gly Lys Thr Ala Phe Asp Ile
Ser Ile Gly Asn Gly Asn Glu Asp Leu 115 120 125Ala Glu Ile Leu Gln
Lys Leu Asn 130 135122103PRTArtificial SequenceSynthetic Construct
122Met Arg Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys1
5 10 15Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg
Glu 20 25 30Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp
Gly Tyr 35 40 45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu
Ile Val Glu 50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
Asp Lys Phe Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn
Gly Asn Glu Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100123103PRTArtificial SequenceSynthetic Construct 123Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu 20 25 30Leu
Ile Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50
55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100124103PRTArtificial SequenceSynthetic Construct 124Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Val Asp Glu Val Arg Glu 20 25 30Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn
100125103PRTArtificial SequenceSynthetic Construct 125Met Arg Gly
Ser His His His His His His Gly Ser Asp Leu Gly Lys1 5 10 15Lys Leu
Leu Glu Ala Ala Arg Ala Gly Gln Val Asp Glu Val Arg Glu 20 25 30Leu
Ile Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr 35 40
45Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu
50 55 60Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Phe
Gly65 70 75 80Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu
Asp Leu Ala 85 90 95Glu Ile Leu Gln Lys Leu Asn 100
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