U.S. patent application number 15/679751 was filed with the patent office on 2018-02-08 for phase transition biopolymers and methods of use.
The applicant listed for this patent is Duke University. Invention is credited to Miriam Amiram, Ashutosh Chilkoti, Felipe Garcia Quiroz.
Application Number | 20180037609 15/679751 |
Document ID | / |
Family ID | 46047982 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180037609 |
Kind Code |
A1 |
Chilkoti; Ashutosh ; et
al. |
February 8, 2018 |
PHASE TRANSITION BIOPOLYMERS AND METHODS OF USE
Abstract
The present disclosure describes environmentally responsive
polypeptides capable of displaying stimuli-triggered conformational
changes in a reversible or irreversible manner that may be
accompanied by aggregation. Polypeptides include a number of
repeated motifs and may be elastomeric or non-elastomeric. The
polypeptides may be used to deliver therapeutics to a biological
site and to develop bioactive polypeptides that are environmentally
responsive.
Inventors: |
Chilkoti; Ashutosh; (Durham,
NC) ; Garcia Quiroz; Felipe; (Durham, NC) ;
Amiram; Miriam; (Chapel Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
46047982 |
Appl. No.: |
15/679751 |
Filed: |
August 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14572391 |
Dec 16, 2014 |
9771396 |
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15679751 |
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13904836 |
May 29, 2013 |
8912310 |
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14572391 |
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13245459 |
Sep 26, 2011 |
8470967 |
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13904836 |
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61386002 |
Sep 24, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 9/5169 20130101; C07K 2319/21 20130101; B82Y 5/00 20130101;
A61K 38/00 20130101; A61P 35/00 20180101; C07K 14/78 20130101; A61K
9/5123 20130101; A61K 8/11 20130101; C07K 19/00 20130101; C07K
14/001 20130101; A61K 47/42 20130101; A61K 8/64 20130101 |
International
Class: |
C07K 14/00 20060101
C07K014/00; A61K 38/00 20060101 A61K038/00; A61K 47/42 20060101
A61K047/42; B82Y 5/00 20060101 B82Y005/00; C07K 19/00 20060101
C07K019/00; A61K 8/11 20060101 A61K008/11; A61K 8/64 20060101
A61K008/64; A61K 9/51 20060101 A61K009/51; C07K 14/78 20060101
C07K014/78 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
no. 5R01-GM61232-08 awarded by the National Institute of Health.
The government has certain rights in this disclosure.
Claims
1. A environmentally responsive polypeptide comprising at least ten
sequences selected from VGAPVG, LGAPVG, VPSALYGVG, VGPAVG, VTPAVG,
VPSDDYGQG, VPSDDYGVG, TPVAVG, VPSTDYGVG, VPAGVG, VPTGVG, VPAGLG,
VPHVG, VHPGVG, VPGAVG, VPGVAG, VRPVG, GRGDSPY, GRGDSPH, GRGDSPV,
GRGDSPYG, RPLGYDS, RPAGYDS, RPXGYDS, GRGDSYP, GRGDSPYQ, GRGNSPYG,
GRGDAPYQ, VPXSRNGG, VPHSRNGG, VPHSRNGL, VPGHSHRDFQPVLHLVALNSPL
SGGMRG, HTHQDFQPVLHLVALNTPLSGGMRGIRPGG, FEWTPGWYQPYG or a
combination thereof, wherein X is from zero to four amino acid
residues, and wherein the polypeptide upon stimulation undergoes a
conformational change that is accompanied by aggregation.
2. The polypeptide of claim 1, wherein the at least ten sequences
are consecutive.
3. The polypeptide of claim 1, further comprising a spacer sequence
between at least two of the at least ten sequences.
4. The polypeptide of claim 3, wherein the spacer sequence
comprises from one to twenty-six amino acids.
5. The polypeptide of claim 1, wherein the polypeptide exhibits
phase separation when exposed to a threshold temperature that is
(i) above a lower critical solution temperature of the polypeptide,
or (ii) below an upper critical solution temperature of the
polypeptide, or exhibits phase separation when exposed to a
threshold temperature that is above the lower critical solution
temperature, and when exposed to a threshold temperature that is
below the upper critical solution temperature.
6. The polypeptide of claim 5, wherein the phase separation is
reversible.
7. The polypeptide of claim 5, wherein the phase separation is
irreversible.
8. The polypeptide of claim 1, wherein the polypeptide exhibits
heat-irreversible phase separation when exposed to a threshold
temperature that is above a lower critical solution temperature of
the polypeptide, and exhibits reversible phase separation below the
threshold temperature.
9. The polypeptide of claim 1, wherein the polypeptide exhibits a
reversible phase separation in response to a first stimulus.
10. The polypeptide of claim 9, wherein the polypeptide exhibits an
irreversible phase separation in response to a second different
stimulus.
11. The polypeptide of claim 1, wherein the at least 10 sequences
convey LCST or UCST transition behavior, and wherein the
polypeptide further comprises at least 9 sequences which are
interspersed among the at least 10 sequences, wherein the at least
9 sequences convey LCST transition behavior when the at least 10
sequences convey UCST transition behavior, and UCST transition
behavior when the at least 10 sequences convey LCST transition
behavior, such that the polypeptide displays both LCST and UCST
transition behavior.
12. A fusion protein comprising the polypeptide of claim 1.
13. A composition comprising the polypeptide of claim 1, conjugated
to a molecule.
14. The composition of claim 13, wherein the molecule is selected
from an oligonucleotide, a therapeutic, a carbohydrate, a synthetic
polymer, or a combination thereof.
15. A polypeptide comprising the at least ten sequences of the
polypeptide of claim 1 as a reverse sequence when read from
C-terminus to the N-terminus.
16. A method of effecting a conformational change in a polypeptide
comprising exposing the polypeptide of claim 1 to a stimulus such
that the polypeptide undergoes a conformational change that is
accompanied by aggregation or solubilization in response to the
stimulus.
17. The method of claim 16, wherein the polypeptide becomes
bioactive or loses bioactivity following the conformational
change.
18. A environmentally responsive polypeptide comprising at least
ten PG motifs, and at least nine spacer sequences between the PG
motifs, the at least nine spacer sequences being between five and
thirty amino acid residues in length and not comprising a PG motif,
and wherein the polypeptide upon stimulation undergoes a
conformational change that is accompanied by aggregation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 14/572,391, filed Dec. 16, 2014, which is a
divisional of U.S. patent application Ser. No. 13/904,836, filed
May 29, 2013, which is a divisional of U.S. patent application Ser.
No. 13/245,459, filed Sep. 26, 2011, which claims priority to U.S.
Provisional Patent Application No. 61/386,002 filed Sep. 24, 2010,
the contents of each of which are incorporated herein by reference
in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 26, 2017, is named
"20917551_028193-9124-US02_As_Filed_Sequence_Listing.txt" and is
40,054 bytes in size.
BACKGROUND
[0004] Elastomeric proteins, and in particular elastin, have been
the subject of extensive investigations aimed at a molecular
understanding of the structure-function relationship among these
proteins, their mechanical properties, environmental sensitivity
and self-assembly properties. These studies indicated a role for
disordered protein structures in proteins and protein domains, and
the identification of short recurrent peptides which were capable
of forming protein-polymers with similar structural and
environmental properties. A general elastic, environmentally
responsive motif Val-Pro-Gly-X-Gly (SEQ ID NO: 135) was found in
elastin, where X is any amino acid except Proline, and was used to
develop elastin-like polypeptides (ELPs) for biotechnological and
biomedical uses. Similarly, resilin-like polypeptides displaying
mechanical properties comparable to the native resilin protein were
made.
[0005] Silks, on the other hand, constitute a complex family of
proteins that encompasses both elastomeric and non-elastomeric
proteins. Elastomeric silks include a highly repetitive GPGGX (SEQ
ID NO: 1) motif. Studies have indicated that there is an absence of
Proline residues in non-elastomeric silks, and that .beta.-sheet
structures increase in proportion to the GPGGX (SEQ ID NO: 1)
content. However, the structures adopted by the abundant GPGGX (SEQ
ID NO: 1)/GPGQQ(SEQ ID NO: 2) and GGX repeat units remain unclear.
Little progress has been made on the design of recombinant
silk-like biomaterials. In addition, elastin remains the only
elastomeric repetitive protein successfully reduced to a short
motif capable of displaying both elasticity and environmental
sensitivity.
[0006] After more than three decades of research since the
discovery of the environmental sensitivity of elastin monomers
(tropoelastin), the more than two decades since the identification
of the canonical elastomeric motif VPGXG (SEQ ID NO: 3), and the
almost two decades since the generalization of this repeat unit
into the canonical ELP motif VPGXG (SEQ ID NO: 3), only a handful
of elastin-inspired polypeptides departing from the canonical
sequence have been uncovered, namely minor modifications of the
canonical motif, such as LPGXG (SEQ ID NO: 4) and IPGXG (SEQ ID NO:
5), and the repeat unit VPAVG (SEQ ID NO: 6). Recent efforts have
made use of complex bioinformatics tools to search for sequence
conservation, amino acid patterns, and recurrent motifs among
elastin proteins from different species; these studies have hinted
at the potential functional role of the PG dipeptide in elastin
(see, e.g, He, D. et al. (2007) Matrix Biology 26:524-540). In a
more general approach, studies of similarities in Proline and
Glycine content between a large panel of elastomeric proteins from
different species including elastin, resilin, gluten, and silks
failed to identify first principles for the design of general
elastomeric motifs, or to identify and reduce to practice novel
motifs responsible for the elasticity and/or environmental
sensitivity of these proteins. (see, e.g., Rauscher, S. et al.
(2006) Structure 14:1667-1676).
SUMMARY
[0007] One aspect of the present disclosure describes
environmentally responsive polypeptides containing at least ten
repeats of an amino acid sequence Z.sub.1Z.sub.2PXGZ.sub.3 (SEQ ID
NO: 22), wherein P is Proline, G is Glycine, X is from 1 to 4 amino
acids that are not Proline or Glycine, and Z.sub.1, Z.sub.2 and,
Z.sub.3 are each an amino acid is described. Upon stimulation the
polypeptide undergoes a conformational change that is accompanied
by aggregation.
[0008] Another aspect describes environmentally responsive
polypeptides that contain at least ten repeats of an amino acid
sequence Z.sub.1PXGZ.sub.2RGZ.sub.3 (SEQ ID NO: 113), wherein P is
proline, G is glycine, R is arginine, D is aspartate, X is from 0
to 4 amino acids that are not proline or glycine, and Z.sub.1 and
Z.sub.2 are each an amino acid, and Z.sub.3 is an amino acid such
as aspartate (D). Upon stimulation the polypeptide undergoes a
conformational change that is accompanied by aggregation.
[0009] Another aspect describes environmentally responsive
polypeptides comprising at least ten sequences selected from VGAPVG
(SEQ ID NO: 24), LGAPVG (SEQ ID NO: 25), VPSALYGVG (SEQ ID NO: 26),
VGPAVG (SEQ ID NO: 17), VTPAVG (SEQ ID NO: 18), VPSDDYGQG (SEQ ID
NO: 29), VPSDDYGVG (SEQ ID NO: 30), TPVAVG (SEQ ID NO: 31),
VPSTDYGVG (SEQ ID NO: 32), VPAGVG (SEQ ID NO: 33), VPTGVG (SEQ ID
NO: 34), VPAGLG (SEQ ID NO: 35), VPHVG (SEQ ID NO: 36), VHPGVG (SEQ
ID NO: 37), VPGAVG (SEQ ID NO: 38), VPGVAG (SEQ ID NO: 39), VRPVG
(SEQ ID NO: 40), GRGDSPY SEQ ID NO: 41), GRGDSPH (SEQ ID NO: 42),
GRGDSPV (SEQ ID NO: 43), GRGDSPYG (SEQ ID NO: 44), RPLGYDS (SEQ ID
NO: 45), RPAGYDS (SEQ ID NO: 46), GRGDSYP (SEQ ID NO: 47), GRGDSPYQ
(SEQ ID NO: 48), GRGNSPYG (SEQ ID NO: 49), GRGDAPYQ (SEQ ID NO:
50), VPHSRNGG (SEQ ID NO: 51), VPHSRNGL (SEQ ID NO: 52),
VPGHSHRDFQPVLHLVALNSPLSGGMRG (SEQ ID NO: 53),
HTHQDFQPVLHLVALNTPLSGGMRGIRPGG (SEQ ID NO: 54), FEWTPGWYQPYG (SEQ
ID NO: 55) or a combination thereof. Upon stimulation the
polypeptide undergoes a conformational change that is accompanied
by aggregation.
[0010] Another aspect describes environmentally responsive
polypeptides that contain at least ten PG motifs, and at least nine
spacer sequences between the PG motifs. The spacer sequences do not
include a PG motif and are between five and thirty amino acid
residues in length. Upon stimulation the polypeptide undergoes a
conformational change that is accompanied by aggregation.
[0011] Another aspect describes an environmentally responsive
polypeptide which upon stimulation undergoes a conformational
change that is accompanied by aggregation and includes at least ten
repeats of an amino acid sequence
Z.sub.1PXGZ.sub.2Z.sub.3Z.sub.4Z.sub.5 (SEQ ID NO: 131), wherein P
is proline, G is glycine, X is from 0 to 4 amino acids that are not
proline or glycine, and Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4 and
Z.sub.5 are each an amino acid. Z.sub.2 may be optionally absent.
The amino acid sequence includes an arginine, as well as either a
serine or aspartate, or both serine and aspartate, and at least one
hydrophobic residue selected from valine, leucine, isoleucine,
histidine, tyrosine, tryptophan and alanine. The asparatate may be
optionally substituted with glutamate.
[0012] Another aspect describes methods of effecting a
conformational change in a polypeptide by exposing the polypeptide
to a stimulus such that the polypeptide undergoes a conformational
change in response to the stimulus. The conformational change may
be accompanied by aggregation or solubilization.
[0013] Another aspect describes nanoparticles formed from an
environmentally responsive polypeptide which may encapsulate a
therapeutic for delivery to a biological site.
[0014] Another aspect describes methods of delivering therapeutics
to biological sites by contacting the biological site with a
nanoparticle formed from an environmentally responsive polypeptide,
wherein the nanoparticle receives a stimulus at the biological site
and disassembles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component is
labeled in every drawing. The foregoing objects, features and
advantages of the present disclosure will become more apparent from
a reading of the following description in connection with the
accompanying drawings in which:
[0016] FIGS. 1A-1C depict graphs showing the distribution of the
VPGXG (SEQ ID NO: 3) motif along the sequence of elastins from
different species: Homo sapiens, Bovine, Zebrafish, Mus musculus
and Rattus (FIGS. 1A and 1B); motifs such as VPGXG (SEQ ID NO: 3),
LPGXG (SEQ ID NO: 4) and IPGXG (SEQ ID NO: 5) altogether account
for only 10-20% of the amino acids in these sequences; sequence
conservation among species is limited. Mapping of minima functional
P(X.sub.n)G motifs along the sequences of Bovine and Homo sapiens
elastin shows predominance of highly conserved PG motifs (i.e.,
sharp peaks) covering the entire sequence as evidenced by abundant
overlapping peaks. FIG. 1C shows the localization of the VPGXG (SEQ
ID NO: 3) motifs as a digital signal where all residues forming
part of the motif have a value of 1 and 0 otherwise; the
P(X.sub.n)G motifs are digitized such that the signal is 1 for
Proline and subsequent residues within a continuous P(X.sub.n)G
motif and 0 for G residues within the motif or any non-motif amino
acid along the sequence.
[0017] FIGS. 2A-2D depict graphs showing the mapping of minima
functional P(X.sub.n)G motifs and corresponding reversed
G(X.sub.n)P motifs along the sequence (only shown for residues
0-250) of Flagelliform silk (FIG. 2A), elastin (FIG. 2B), gluten
(FIG. 2C) and resilin (FIG. 2D). Similar analyses were performed
for other proteins listed in Table 2. The directionality of the
motif is evidenced for all proteins, except Flagelliform silk (and
Dragline silk, not shown here), where GP and PG motifs (i.e., sharp
red and blue peaks, respectively) show the same distribution as a
result of the abundant GPGXX (SEQ ID NO: 7) motif, which displays
both minima motifs. Increasing frequency of P(X.sub.n)G motifs with
larger n values is observed when comparing the elastomeric proteins
Flagelliform silk, gluten, elastin and resilin (FIGS. 2A-2D).
[0018] FIGS. 3A-3E depict graphs showing quantitation of minima
functional motifs among different elastomeric and non-elastomeric
proteins. Occurrence of P(X.sub.n)G motifs before (FIG. 3A) and
after (FIG. 3B) normalizing to the total length of the protein.
FIG. 3C shows quantitation of VPG, VPGXG (canonical ELP motif; SEQ
ID NO: 3) and PG motifs in elastin from different species. FIG. 3D
shows the percentage of P(X.sub.n)G motifs preceded by Glycine to
yield GP(X.sub.n)G motifs, as shown for n=0, n=1, n=2 and
n.gtoreq.3 (indicated as PGX.sub.nG in the figure). FIG. 3E shows
the average distance between P(X.sub.n)G motifs with either n=0 or
n=1 compared with average distance between a control P(X.sub.n)G
motif
[0019] FIG. 4 is a graph showing the hydropathy index (Hi)
distribution for residues surrounding P(X.sub.n)G motifs (n=0) in
elastin sequences from Homo sapiens, Bovine, Zebrafish and Mus
musculus. This analysis considers residues one position before
(Pm1, for "Proline minus 1 residue") and 2 positions before (Pm2) a
Proline residue in a P(X.sub.n)G motif, as well as residues one
position after (Gp1, for "Gly plus 1 residue") and two positions
after (Gp2) a Gly residue in a P(X.sub.n)G motif. The Hi was
defined as proposed by Kyte and Doolittle (1982). The identity of
the residues at a given position can be easily read from this
figure (e.g., Gly=-0.4; Val=4.2; A=1.8, etc.). The box delineates
the 25 and 95 percentile, and the mean hydropathy is indicated by a
square; raw data is also included for each residue position as
filled diamonds.
[0020] FIG. 5 is a graph showing hydropathy index (Hi) distribution
for residues surrounding P(X.sub.n)G motifs (n=0) in elastin (Homo
sapiens), resilin, gluten, collagen (type II.alpha.1, Col2A1),
fibronectin, titin, Flagelliform and Dragline silks. This analysis
considers residues one position before (Pm1, for "Proline minus 1
residue") and 2 positions before (Pm2) a Proline residue on a
P(X.sub.n)G motif, as well as residues one position after (Gp1, for
"Gly plus 1 residue") and two positions after (Gp2) a Gly residue
in a P(X.sub.n)G motif The Hi was defined as proposed by Kyte and
Doolittle (1982). The identity of the residues at a given position
can be easily read from this figure (e.g., Gly=-0.4; Val=4.2;
A=1.8, etc.). The box delineates the 25 and 95 percentile, and the
mean hydropathy is indicated by a square; raw data is also included
for each residue position as filled diamonds.
[0021] FIG. 6 is a graph showing hydropathy index (Hi) distribution
for residues comprising and surrounding P(X.sub.n)G motifs (n=1) in
collagen. In addition to Pm2, Pm1, Gp1 and Gp2, this analysis
considers the residue Pp1 (for "Pro plus 1 position"), which is
equivalent to the X residue constituting the P(X.sub.n)G motif The
Hi was defined as proposed by Kyte and Doolittle (1982). The
identity of the residues at a given position can be easily read
from this figure (e.g., Gly=-0.4; Val=4.2; A=1.8, etc.). The box
delineates the 25 and 95 percentile, and the mean hydropathy is
indicated by a square, raw data is also included for each residue
position as filled diamonds.
[0022] FIG. 7 is a graph showing hydropathy index (Hi) distribution
for residues comprising and surrounding P(X.sub.n)G motifs (n=4) in
resilin and titin. Whereas resilin shows a very tight Hi
distribution in all positions comprising and surrounding the
P(X.sub.4)G motif, accounting for 29.5% of the full protein
sequence, the same motif accounts for only 1.6% of the amino acid
sequence of titin, which was used for comparison due to its
elasticity and remarkable length (26916 residues). Fibronectin and
Col2A1 (Homo sapiens) have 0.12% and 0%, respectively. Titin shows
a broad distribution of Hi indices, with the exception of Gp1 where
only Gly occurs. In addition to Pm2, Pm1, Gp1 and Gp2, this
analysis considers the residues Pp1 (for "Pro plus 1 position"),
Pp2, Pp3, Pp4, which are equivalent to the 4.times. residues
constituting the P(X.sub.n)G motif The Hi was defined as proposed
by Kyte and Doolittle (1982). The identity of the residues at a
given position can be easily read from this figure (e.g., Glu=-0.4;
Val=4.2; A=1.8, etc.). The box delineates the 25 and 95 percentile,
and the men hydropathy is indicated by a square; raw data is also
included for each residue position as filled diamonds.
[0023] FIGS. 8A and 8B depict graphs showing environmental
sensitivities of EIPs with Z.sub.1Z.sub.2PGZ.sub.3Z.sub.4 (SEQ ID
NO: 9) motif, exemplified for both hexapeptide (FIG. 8A) and
pentapeptide (FIG. 8B) repeat units. This family of EIPs displays
inverse phase transition behavior characterized by a sharp phase
separation and self-assembly upon heating above the LCST or Tt of
these polypeptides. The number of repeats in each protein-polymer
is indicated in the legend. All samples were prepared at a
concentration of 50 .mu.M in PBS, with the exception of (APGVGP
(SEQ ID NO: 132)) and (TVPGAG (SEQ ID NO: 10)) that were diluted in
PBS supplemented with 2M NaCl, at 50 and 100 .mu.M,
respectively.
[0024] FIGS. 9A-9D depict graphs and a photograph showing the
characterization of a library of EIPs with the AVPGVG (SEQ ID NO:
8) repeat. Turbidity profiles for 5 constructs in this library at
25 .mu.M in PBS (FIG. 9A) and PBS supplemented with 1M NaCl (FIG.
9B). The transition temperatures calculated from (FIG. 9B) varied
linearly as the reciprocal of molecular weight of the EIP as
expected for canonical ELP sequences (FIG. 9C). The distribution of
molecular weights in this library is illustrated in (FIG. 9D),
where EIP-MW1 corresponds to the lowest and EIP-MW5 to the highest
molecular weight.
[0025] FIGS. 10A-10B depict graphs showing reversible phase
transition behavior displayed by EIPs with
Z.sub.1Z.sub.2PGZ.sub.3Z.sub.4 (SEQ ID NO: 9) motif This family of
EIPs displays reversible inverse phase transition behavior
characterized by a sharp phase separation and self-assembly upon
heating above the LCST or Tt of these polypeptides, followed by
disassembly upon lowering the temperature below the LCST (FIG.
10A). Furthermore, upon heating in a second heating cycle (FIG.
10B), these EIPs retain their environmental sensitivity. The number
of repeats in each protein-polymer is indicated in the legend. All
samples were prepared at a concentration of 50 .mu.M in PBS, with
the exception of (TVPGAG; SEQ ID NO: 10) that was diluted to 100
.mu.M in PBS supplemented with 2M NaCl.
[0026] FIGS. 11A-11D depict graphs showing reversible and
heat-irreversible phase transition behavior displayed by some EIPs
with Z.sub.1Z.sub.2PGZ.sub.3Z.sub.4 (SEQ ID NO: 9) motif This
family of EIPs displays inverse phase transition behavior
characterized by a sharp phase separation and self-assembly upon
heating above their LCST or Tt, which may be reversible or
irreversible for temperatures below or above a threshold
temperature (FIGS. 11A-11D), and which may also be concentration
dependent (FIG. 11C). The number of repeats in each protein-polymer
is shown in the legend. Unless indicated in the legend, the samples
were prepared at 50 .mu.M in PBS.
[0027] FIGS. 12A-12B depict graphs showing results of environmental
sensitivity of EIPs with Z.sub.1Z.sub.2PGZ.sub.3Z.sub.4 (SEQ ID NO:
9) motif, exemplified for both hexapeptide (FIG. 12A) and
pentapeptide (FIG. 12B) repeat units. This family of EIPs displays
inverse phase transition behavior characterized by a sharp phase
separation and self-assembly upon heating above the LCST or Tt of
each polypeptide. The number of repeats in each protein-polymer is
indicated in the legend. All samples were prepared at a
concentration of 50 .mu.M in PBS, with the exception of the
randomized EIP ([ZZPXGZ).sub.5]-.sub.4) [(SEQ ID NO:
11).sub.5]-.sub.4 with sequence
(GAPFGFAIPMGAGFPTGGLAPFGMGLPAGM).sub.4, (SEQ ID NO: 12).sub.4 which
was prepared in PBS with 6 M Urea and 1M NaCl.
[0028] FIGS. 13A-13F depict graphs showing the reversible and
heat-irreversible phase transition behavior displayed by different
EIPs with Z.sub.1Z.sub.2PX.sub.1GZ.sub.3Z.sub.4 (SEQ ID NO: 13)
motif. This family of EIPs displays inverse phase transition
behavior characterized by a sharp phase separation and
self-assembly upon heating above the polypeptides LCST or Tt, which
may be reversible (FIG. 11A, FIG. 11B, FIG. 11D, FIG. 11E), or
irreversible (FIG. 11C, FIG. 11F) for temperatures above a
threshold temperature or for buffer ionic strengths above a
threshold ionic strength (FIG. 11E). The number of repeats in each
protein-polymer is shown in the legend. Unless indicated in the
legend, the samples were prepared in PBS at 50 .mu.M.
[0029] FIG. 14 is a graph showing results of environmental
sensitivity of EIPs with
Z.sub.1Z.sub.2PX.sub.1X.sub.2GZ.sub.3Z.sub.4 (SEQ ID NO: 14) motif,
exemplified for both hexapeptide and pentapeptide repeat units.
This family of EIPs displays inverse phase transition behavior
characterized by a sharp phase separation and self-assembly upon
heating above the LCST or Tt of each polypeptide. The number of
repeats in each protein-polymer is indicated in the legend. All
samples were prepared at a concentration of 50 .mu.M in PBS.
[0030] FIGS. 15A-15D depict graphs showing the reversible and
heat-irreversible phase transition behavior displayed by different
EIPs with Z.sub.1Z.sub.2PX.sub.1X.sub.2GZ.sub.3Z.sub.4 (SEQ ID NO:
14) motif. This family of EIPs displays inverse phase transition
behavior characterized by a sharp phase separation and
self-assembly upon heating above the polypeptide LCST or Tt, which
may be reversible (FIG. 15A, FIG. 15B), or irreversible (FIG. 15C,
FIG. 15D) for temperatures above a threshold temperature. The
number of repeats in each protein-polymer is shown in the legend.
Unless indicated in the legend, the samples were prepared in PBS at
50 .mu.M.
[0031] FIGS. 16A-16B depict graphs showing reversible phase
transition behavior by an EIP with
Z.sub.1Z.sub.2PX.sub.1X.sub.2X.sub.3X.sub.4GZ.sub.3Z.sub.4 (SEQ ID
NO: 15) motif. This EIP displays reversible inverse phase
transition behavior characterized by a sharp phase separation and
self-assembly upon heating above its LCST or Tt, followed by
disassembly upon lowering the temperature below the LCST (FIG.
16A-16B). Furthermore, upon heating in a second heating cycle (FIG.
16A-16B), this EIP retains its environmental sensitivity. Samples
in (FIG. 16A) were prepared at 25 .mu.M in PBS and 8 M Urea, pH 7
(PBSU), and samples in (FIG. 16B) were prepared in PBS 8 M Urea at
pH 9 at the indicated concentrations.
[0032] FIGS. 17A-17D depict graphs showing retro-EIPs display
thermoresponsive behavior distinct to the observed in the parent
EIP (shown in red) despite having the same hydrophobicity profiles
and amino acid side-chain relationships. Occurrence of Gly at Pm1
(GP motif) upon backbone reversal resulted in a pronounced decrease
in the Tt and the emergence of heat-irreversible phase separation
upon heating above a threshold temperature (FIG. 17A-17B).
Noteworthy, the difference in the Tt observed in (FIG. 17A) is
underestimated, as the Tt of (VPGVG)30 (SEQ ID NO: 97).sub.30 is
likely to be .about.10.degree. C. higher than shown, since this
construct lacks a His-tag present in (VGPVG)30 (SEQ ID NO:
98).sub.30; then, the overall .DELTA.Tt in (FIG. 17A) is
.about.30.degree. C., which is likely to be similar to the
.DELTA.Tt observed in (FIG. 17B). Retro-EIP in (FIG. 17C), wherein
the parent PX1G motif was conserved, suggests that overall
hydrophobicity is a good predictor of the inverse transition
temperature of Z1Z2PX1GZ1Z2 (SEQ ID NO: 13) motifs. The large
hysteresis displayed by VPAVG (SEQ ID NO: 6) (See FIG. 15A), is
absent in the corresponding retro-EIP (FIG. 17D). Samples were
prepared in PBS at 50 .mu.M unless indicated.
[0033] FIG. 18A-18C depict graphs and photographs showing modified
retro-EIPs unravel the role of Gly at Pm1 in the environmental
sensitivity and self-assembly properties of EIPs comprised of
minima functional PX.sub.nG motifs. Substituting a hydrophobic
residue (relative to Gly) for Gly residue at Pm1, results in an
unexpected dramatic increase in the Tt of the modified retro-EIP
(shown in red), which restores the transition temperature to a
value closer to that of the parent EIP (FIGS. 17A-B) and the
sensitivity to changes in buffer ionic strength (FIG. 18B), and
suppresses heat-irreversible phase transition behavior (FIG. 18A).
(FIG. 18C) Phase contrast microscopic analysis of the structures
formed upon self-assembly of EIP constructs drying on glass
surfaces. (Ca) Self-assembled fractals formed by EIP (VPAVG).sub.45
(SEQ ID NO: 6).sub.45 with a PX.sub.1X.sub.2G (SEQ ID NO: 16)
minima functional motif (Cb) Retro-EIP (VGPAVG).sub.20 (SEQ ID NO:
17).sub.20 where Pm1 is Gly does not form fractal structures and
assembles into fibrillar-like densely packed structures. (Cc) The
modified retro-EIP (VTPAVG).sub.20 (SEQ ID NO: 18).sub.20 shows
restored self-assembly into fractal structures. (Cd) Upon
rehydration of the imaged droplets. Large fibrils are observed for
the retro-EIP (Cd), while only small submicron aggregates
(evidenced in the roughness of the surface) are observed for the
modified retro-EIP (Ce). (Cf) Environmental scanning electron
microscopy shows similar fractal structures formed by an
elastin-like polypeptide (with motif VPGXG (SEQ ID NO: 3), where
X=[A:G]) when frozen-dried above its transition temperature. Scale
bar is 50 .mu.M. Note that threonine is more hydrophobic than Gly
according to Urry's hydrophobicity scale (see, Urry, D. W. et al.
(1992) Biopolymers 32:1243-1250).
[0034] FIG. 19 is a graph showing self-assembly of EIP with
sequence (VTPAVG)20 (SEQ ID NO: 18).sub.20, comprised of a minima
functional motif PX1X2G (SEQ ID NO: 16), into nanostructures. The
formation of a third population (P3) (.about.4% mass), with
hydrodynamic radius larger than 1 mm, for temperatures above 38oC,
reduced the quality of the DLS data and prevented data acquisition
at higher temperatures. Noteworthy, turbidity profiles demonstrate
stable light scattering properties for temperature between 40oC and
75oC, presumably arising from small micrometer sized particles;
major aggregation is expected to occur at .about.80oC, considering
that major aggregation of (VPTAVG)25 ((SEQ ID NO: 18)25) occurs at
.about.72.degree. C. (FIG. 18B). Sample was prepared at 50 .mu.M in
PBS.
[0035] FIG. 20 is a graph showing thermoresponsive behavior of
self-cross-linkable in situ gelling materials composed of
elastomeric-inspired polypeptides. Cysteine residues were
incorporated into EIPs displaying PG and PX1G motifs to enable
their self-cross-linking via disulfide bonding. The primary
sequence of these protein polymers is described as (ZVPGXG).sub.144
((SEQ ID NO: 20).sub.144) and (VPZGXG).sub.144 ((SEQ ID NO:
19).sub.144), where [Z,X][5A:1C, V], that is 1 Cys every 5 Ala
residues in the Z position, and a Cal in the X position for every
repeat, for a total of 144 repeat units. Noteworthy, the EIP
(ZVPGXG)144 ((SEQ ID NO: 6 20)144)was designed to display the
bioactive motif GXXPG (SEQ ID NO: 21) responsible for elastin
bioactivity. The inverse transition temperature of these
self-cross-linkable EIPs was engineered to occur below body
temperature to allow for rapid coacervation before the onset of
gelation via disulfide bonding.
[0036] FIGS. 21A-21F depict graphs showing multiple Pro and Gly
arrangements besides the canonical Pro-Gly dipeptide are conducive
to unstructured protein-polymers that display "smart" behavior.
Protein-polymers with periodic Pro and Gly residues arranged as
PX.sub.nG units, where n=0 (FIG. 21A), 1 (FIG. 21B), 2 (FIG. 21D),
3 and 4 (FIG. 21E), and having pentapeptide, hexapeptide and
nonapeptide repeat units display thermally-triggered phase
transition behavior. These protein-polymers lack ordered secondary
structures as shown by their circular dichroism spectra
characteristic of disordered proteins (FIG. 21C). We found that
protein-polymers in FIGS. 21A, 21B, 21D and 21E fall within any of
three types of phase transition behavior, corresponding to three
degrees of hysteresis in the reversibility of their
thermally-triggered phase transition: zero, finite and
heat-sensitive, infinite hysteresis (FIG. 21F). All turbidity
measurements were conducted in PBS at a polypeptide concentration
of 50 .mu.M, except for VRPVG (SEQ ID NO: 40) (+1M NaCl), VAPGVG
(SEQ ID NO: 67) (+0.5 M NaCl), APGVG (SEQ ID NO: 99) (+2 M NaCl)
and VPSALYGVG (SEQ ID NO: 26) (+8 M urea). CD studies were
conducted in water at a polypeptide concentration of 5 .mu.M.
[0037] FIGS. 22A-22K depict graphs showing that "smart" biopolymers
exhibit protein-like features. A simple method (FIG. 22A) was
applied which was inspired in the do's and don'ts of non-fibrillar
Pro and Gly-rich proteins to favor protein disorder, to engineer a
protein-sized, 240-residue-long proteinpolymer with a target
hydropathy--equivalent to transition temperature in of 37.degree.
C. and composed of 40 hexapeptide motifs containing Pro-X-Gly units
with randomly selected amino acids spanning a broad range of
hydropathies (FIG. 22B). This protein-sized biopolymer behaved as
an intrinsically disordered protein (FIG. 22C) and exhibited
"smart", phase transition behavior (FIG. 22D). Gly residues
preceding a PXnG unit modulated the phase transition behavior of
"smart" proteinpolymers (FIGS. 22E-22F). The inset of (FIG. 22F)
shows the thermally-triggered assembly of a n environmentally
responsive polypeptide with repeat unit VTPAVG (SEQ ID NO: 28) that
was not observed for the Gly-variant VGPAVG (SEQ ID NO: 17). (FIG.
22G) Backbone-reversed protein-polymers present identical amino
acid patterns, which were exemplified with the structure of a
"smart" pentapeptide motif and its retro-motif as observed in the
crystal structure of two different 7 proteins (PDB id 3MKR_B and
1OZP, respectively). Changes in the phase behavior of "smart"
protein-polymers on backbone-reversal (FIGS. 22H-22I, Fig S11A)
were observed--mostly changes in the hysteresis of the
transition--as well as large changes in the ensemble of dynamic
conformers that characterize their secondary structure (FIG. 22K,
Fig S11B-D).
[0038] FIGS. 23A-23G depict graphs showing environmentally
responsive polypeptides having a syntax that is truly protein-like.
First, two protein-polymers were designed with identical amino acid
composition (FIG. 23A), "smart" matrikine 1 (SM1) and SM2, where
only SM1 conforms to a bioactive motif GXXPG (SEQ ID NO: 21) found
in various extracellular matrix proteins. These materials were
engineered to self-gel upon subcutaneous injection by enabling the
formation of disulfide bonds upon phase transition through
carefully spaced Cys residues. (FIG. 23B) SM1 and SM2 displayed
identical phase transition behavior in PBS (pH 7.4) and coacervated
below body temperature. (FIG. 23C) The bioactive SM1(at 350 .mu.M
in PBS) prevented tumor growth when used as vehicle for the
inoculation of 0.5.times.105 HT-1080 tumor cells into the leg
(n=10) of nude mice, whereas the non-bioactive SM2 (at 350 .mu.M in
PBS) had no effect on tumor growth. Asterisk (*) indicates
statistical significance with a 95% confidence using a Bonferroni
test to compare the mean tumor volumes after 17 days of tumor
inoculation. FIG. 27 shows the anti-tumor activity of SM1-24 for
tumors inoculated in the back of nude mice. In a second example,
the complex and long peptide sequences forming the bioactive sites
of murine (PDB file: 1DY0) and human endostatin (PDB file: 1BNL)
(FIG. 23D), which amino acid sequences are shown in blue and red,
respectively, are conferred with "smart" behavior on
polymerization. These environmentally responsive polypeptides
behave as intrinsically disordered proteins and are highly stable
in aqueous solution (FIG. 23E), and display inverse phase
transition behavior (FIG. 38) (FIG. 23F). The ability to design
"smart" protein-polymers with monomer units that have defined,
local secondary structure propensities, as in human and murine
endostatin (FIG. 23G, within the box), may enable the development
of a broader set of "smart", drug-like protein-polymers derived
from the growing list of polypeptide hormones that remain partially
disordered on polymerization (FIG. 23G). Additional peptide
hormones of interest were studied. Secondary structures were
predicted using the Jnet algorithm, where `H` is .alpha.-helix, `E`
is .beta.-sheet, and `-` is random coil. Circular dichroism data
were obtained in water at a polypeptide concentration of 5 .mu.M.
Images of the 3D structures of endostatin were rendered using
PyMOL.
[0039] FIG. 24 is a graph showing environmentally responsive
"smart" behavior of a polypeptide with a non-repetitive, 36 amino
acid long repeat unit, designed as in FIG. 22A, but using a target
hydropathy of 2, according to Kyte-Dolittle's scale.
[0040] FIG. 25 depicts graphs showing that high Gly content is not
a prerequisite for the design of environmentally responsive
polypeptides. Gly residues are not a sine qua non element for the
design of protein-polymers that display fully reversible phase
transition behavior. Protein-polymer concentration was 50 .mu.M
unless otherwise indicated.
[0041] FIGS. 26A-26D depict graphs showing backbone reversal of an
environmentally responsive polypeptide results in pronounced
changes in phase behavior and secondary structure propensities.
(FIG. 26A) Changes in phase behavior for a fourth test-in-case of
the effect of backbone reversal on phase behavior. The
retro-polypeptide showed a greater propensity for coacervation but
reduced sensitivity to ionic strength. (FIGS. 26C-26D) The circular
dichroism spectra of three pairs of environmentally responsive
polypeptides and their respective retro-motifs revealed significant
changes in the overall disorder of the structures (negative peak at
.about.200 nm) and .beta.-turn content (negative region around 210
nm). Turbidity studies were conducted in PBS (with salt
concentrations as indicated) at a polypeptide concentration of 50
.mu.M. CD studies were done in water at a polypeptide concentration
of 5 .mu.M.
[0042] FIG. 27 is a graph showing that a polypeptide containing a
matrikine motif GXXPG, SM1-24 (250 .mu.M in PBS), prevented the
grafting of 1.times.10.sup.6 HT1080 tumor cells inoculated into the
back of nude mice. A control polypeptide, SM2-24 (250 .mu.M in
PBS), with a disrupted motif but identical phase transition
behavior (FIG. 23) had no effect on tumor growth. Tumor volumes
were measured 19 days after inoculation.
[0043] FIG. 28 depicts a graph and photograph showing that
environmentally responsive polypeptides may be designed to display
UCST behavior.
[0044] FIG. 29 is a graph showing reversible UCST behavior in PBS
which behavior may be tuned by polypeptide concentration and the
number of repeating units.
[0045] FIG. 30 is a graph showing bioactive environmentally
responsive polypeptides incorporating the peptide drug GRGDSP (SEQ
ID NO: 133).
[0046] FIG. 31. is a graph showing the UCST behavior of
environmentally responsive polypeptides containing RGD
tripeptides.
[0047] FIG. 32 is a graph showing the UCST behavior of
environmentally responsive polypeptides may be modulated by
electrostatic interactions between positively and negatively
charged amino acids within the sequence.
[0048] FIG. 33 is a graph showing the UCST behavior of
environmentally responsive polypeptides does not require
electrostatic interactions.
[0049] FIG. 34 is a graph showing that RGD-containing
environmentally responsive polypeptides that display UCST behavior
are compatible with multiple arrangements of Pro and Gly
residues.
[0050] FIG. 35 is a graph showing that the UCST behavior of
environmentally responsive polypeptides can be tuned by adjusting
the hydrophobicity of the residues comprising the repeating
unit.
[0051] FIG. 36 is a graph showing that environmentally responsive
polypeptides that contain the peptide drug PHSRN (SEQ ID NO: 107)
display UCST behavior.
[0052] FIG. 37 is a graph showing that environmentally responsive
polypeptides may be designed to display complex phase
behaviors.
[0053] FIGS. 38A-38B are grafts showing that an environmentally
responsive polypeptide based on the bioactive site of murine
Endostatin displayed an inverse phase transition temperature
reminiscent of other environmentally responsive polypeptides with
simpler syntax. The phase transition of a 5 .mu.M solution of
mEndo1-6 in PBS (pH 6.4) (FIG. 38A) was accompanied by a decrease
in the disorder of the polypeptide conformation (FIG. 38B), as
measured by circular dichroism under identical conditions as in
(FIG. 38A).
DETAILED DESCRIPTION
[0054] The present disclosure describes a model for the design of
elastomeric and non-elastomeric protein-polymers and polypeptides
which are environmentally responsive, by introducing repeats of
functional motifs of the form Z.sub.1Z.sub.2PXGZ.sub.3 (SEQ ID NO:
22) or Z.sub.1Z.sub.2PXGZ.sub.3Z.sub.4 (SEQ ID NO: 22), wherein in
each case P is Proline, G is Glycine, X is from 1 to 4 amino acids
that are not Proline or Glycine, and Z.sub.1, Z.sub.2, Z.sub.3 and
Z.sub.4 are any amino acid. In certain embodiments, Z.sub.1,
Z.sub.2, Z.sub.3 and Z.sub.4 do not generate a PG (proline-glycine)
motif The present disclosure also describes environmentally
responsive polypeptides which include ten or more sequences
selected from VGAPVG (SEQ ID NO: 24), LGAPVG (SEQ ID NO: 25),
VPSALYGVG (SEQ ID NO: 26), VGPAVG (SEQ ID NO: 27), VTPAVG (SEQ ID
NO: 28), VPSDDYGQG (SEQ ID NO: 29), VPSDDYGVG (SEQ ID NO: 30),
TPVAVG (SEQ ID NO: 31), VPSTDYGVG (SEQ ID NO: 32), VPAGVG (SEQ ID
NO: 33), VPTGVG (SEQ ID NO: 34), VPAGLG (SEQ ID NO: 35), VPHVG (SEQ
ID NO: 36), VHPGVG (SEQ ID NO: 37), VPGAVG (SEQ ID NO: 38), VPGVAG
(SEQ ID NO: 39), VRPVG (SEQ ID NO: 40), GRGDSPY (SEQ ID NO: 41),
GRGDSPH (SEQ ID NO: 42), GRGDSPV (SEQ ID NO: 43), GRGDSPYG (SEQ ID
NO: 44), RPLGYDS (SEQ ID NO: 45), RPAGYDS (SEQ ID NO: 46), GRGDSYP
(SEQ ID NO: 47), GRGDSPYQ (SEQ ID NO: 48), GRGNSPYG (SEQ ID NO:
49), GRGDAPYQ (SEQ ID NO: 50), VPHSRNGG (SEQ ID NO: 51), VPHSRNGL
(SEQ ID NO: 52), VPGHSHRDFQPVLHLVALNSPLSGGMRG (SEQ ID NO: 53),
HTHQDFQPVLHLVALNTPLSGGMRGIRPGG (SEQ ID NO: 54), FEWTPGWYQPYG (SEQ
ID NO: 55), or any combination thereof.
[0055] Environmentally responsive polypeptides including the motif
(Z.sub.1Z.sub.2PGZ.sub.3G).sub.n (SEQ ID NO: 56).sub.n may also be
formed, where Z.sub.1, Z.sub.2 and Z.sub.3 are any amino acid.
These polypeptides may be bioactive, elastic or a combination
thereof. This motif, when repeated consecutively, includes the
bioactive motif GXXPG (SEQ ID NO: 21) responsible for elastin's
ability to control various physiological processes including
inflammation, chemotaxis, cell proliferation and differentiation,
extracellular matrix remodeling and the like. Polypeptides
containing the motif (Z.sub.1Z.sub.2PGZ.sub.3G).sub.n (SEQ ID NO:
56).sub.n may be elastic and environmentally responsive and provide
a bioactive signal when used to form recombinant elastin-like
materials.
[0056] Also described are polypeptides in which the cell adhesion
peptide GRGDSP (SEQ ID NO: 133) is modified to conform to a P(Xn)G
motif The phase transition temperature of a polypeptide containing
such a motif may be controlled by adding or modifying residues
incorporated into the motif, such that the original cell adhesion
signal is modulated to signal, for example, exclusively through
integrins or through both integrins and the elastin-binding
receptor. Examples of such polypeptides include those containing
the octapeptides (GRGDSPZG).sub.n ((SEQ ID NO: 57).sub.n) and
(GRGDSPGZ).sub.n ((SEQ ID NO: 58).sub.n), and FEWTPGWYQPY (SEQ ID
NO: 59). Environmentally responsive polypeptides may include one or
more of a PG motif, PX1G motif (where X.sub.1 is an amino acid), or
combination thereof.
[0057] The protein polymers produced are environmentally
responsive, and may be elastomeric or non-elastomeric. The present
disclosure demonstrates minima functional motifs that confer
environmental responsiveness to polypeptides thereof. The
environmental sensitivity may be tuned by varying polypeptide
molecular weight, polypeptide concentration, buffer ionic strength,
hydrophobicity of amino acids in non-essential positions within the
motif, the number of residues separating Pro and Gly, and the
precise localization of additional Gly residues which may surround
the P(X.sub.n)G unit. Polypeptides incorporating the motifs may be
elastomeric or non-elastomeric protein-polymers, and may display
reversible inverse phase transition behavior and/or
heat-irreversible inverse phase transition above a critical
temperature, typically higher than body temperature.
[0058] Environmentally responsive refers to the property of a given
polypeptide to undergo conformational changes, such as coacervation
or aggregation, in response to an external stimulus. Aggregation
may be reversible or irreversible. The environmentally responsive
polypeptide may respond to a small change in stimulus with a
pronounced physical change in one or more properties, such as a
sharp change in solubility. Without limiting the scope of this
disclosure, examples of stimuli include changes in temperature, pH,
chemicals, electric field, and buffer ionic strength.
[0059] Polypeptides described herein may undergo a reversible phase
transition or an irreversible soluble-to-insoluble phase transition
in aqueous solution upon heating through a characteristic
transition temperature (lower critical phase transition or LCST).
If reversible, the transition temperature at which the polypeptide
resolubilizes and transitions from insoluble-to-soluble may be the
same as, or different from the soluble-to-insoluble phase
transition temperature. Polypeptides may exhibit phase separation
when exposed to a threshold temperature that is above a lower
critical solution temperature (LCST) of the polypeptide, or may
exhibit phase separation when exposed to temperatures below an
upper critical solution temperature (UCST) of the polypeptide.
Polypeptides described herein may also exhibit phase separation
when exposed to threshold temperatures both above the lower
critical solution temperature (LCST) and below the upper critical
solution temperature (UCST). Phase separation may reversible or
irreversible. In certain embodiments, a polypeptide may exhibit a
reversible phase separation in response to a one stimulus and an
irreversible phase separation in response to a different stimulus.
The different stimulus may be different in type or degree.
[0060] The difference between the two transition temperatures may
be at least about 1.degree. C., at least about 2.degree. C., at
least about 3.degree. C., at least about 4.degree. C., at least
about 5.degree. C., at least about 6.degree. C., at least about
7.degree. C., at least about 8.degree. C., at least about 9.degree.
C., at least about 10.degree. C., at least about 12.degree. C., or
at least about 15.degree. C. The resolubilization transition
temperature may be higher or lower than the soluble-to-insoluble
phase transition temperature. The polypeptides may undergo inverse
temperature transition, becoming more ordered as the temperature
increases.
[0061] The compositions and polypeptides described herein display a
surprising environmentally responsive profile. For example, it has
been alleged that the pentapeptide motif VPXVG displays inverse
temperature transition only if X=A, where A is Alanine (see, e.g.,
Bessa, P. C. et al. (2010; J Control Release. 142(3):312-8). The
inventors surprisingly found that this was not the case. The
present disclosure introduces functional motifs that facilitate
formation of stable, ordered, secondary structures in a given
dynamic range of stimuli sensed by the polypeptides (e.g., below
the LCST of a polypeptide, or the UCST of a polypeptide).
Furthermore, the present disclosure describes elastomeric and/or
environmentally responsive polypeptides which are protein polymers
containing pentapeptide, hexapeptide, septaheptide, octapeptide,
nonapeptide motifs, or a combination thereof. The motifs may be at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least
15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, at least
25, at least 26, at least 27, at least 28, at least 29, at least
30, at least 35, at least 40, at least 45, at least 50, at least
55, at least 60, at least 65, at least 70, at least 75, at least
80, at least 85, at least 90, at least 95, at least 100, at least
110, at least 120, at least 130, at least 140, or at least 150
amino acids in length. Suitable amino acids include naturally
occurring amino acids, D-amino acids, L-amino acids, and
synthesized amino acids, such as unnatural amino acids. Table 1
displays the transition behavior of certain exemplary motifs
disclosed herein, with respect to displaying UCST, LCST or
both.
TABLE-US-00001 TABLE 1 LCST and USCT Behavior of Exemplary
Polypeptide Motifs Transition Sequence Motifs Behavior VAPVG (SEQ
ID NO: 27) LCST VGAPVG (SEQ ID NO: 24) LCST LGAPVG (SEQ ID NO: 25)
VPSALYGVG (SEQ ID NO: 26) LCST VGPAVG (SEQ ID NO: 17) LCST VTPAVG
(SEQ ID NO: 18) LCST TVPGAG (SEQ ID NO: 71) LCST AVPGVG (SEQ ID NO:
8) TVPGVG (SEQ ID NO: 70) AVPGVGAVPGVGAVPGVGAVPGVGAVPGVGCVPG VG
(SEQ ID NO: 64) GAPFGFAIPMGAGFPTGGLAPFGMGLPAGM LCST (SEQ ID NO: 12)
VPSDDYGQG (SEQ ID NO: 29) LCST and UCST VPSDDYGVG (SEQ ID NO: 30)
LCST TPVAVG (SEQ ID NO: 31) LCST VPSTDYGVG (SEQ ID NO: 32) LCST
VPAGVG (SEQ ID NO: 33) LCST VPTGVG (SEQ ID NO: 34) (for each)
VPAGLG (SEQ ID NO: 35) VPAGVGVPAGVGVPAGVGVPAGVGVPAGVGVPCG VG (SEQ
ID NO: 65) GVPAGHRYPIGGGQPHGKGCPDGVFRPVGLGAPYG
HGAPNGMHRPLGIGKPRGHMYPKGQGQPMGHLVP
DGVGFPRGRKKPVGVGKPIGNGHPIGARTPLGYGM
PDGVGMPMGLFLPNGHGAPHGQGYPAGKLIPKGKG
HPFGKGRPLGAGRPTGFKMPKGLGKPMGVGQPQG
HFVPFGLGQPTGQGAPRGGSQPAGLGHPLGAGAPA
GRCHPYGMGVPRGLAMPRGHGQPRGVGYPKGH (positions 5-244 of SEQ ID NO:
105) GVGPAGHRYPIGGQGPHGKCGPDGVFRPVGLAGPY
GHAGPNGMHRPLGIKGPRGHMYPKGQQGPMGHLV
PDGVFGPRGRKKPVGVKGPIGNHGPIGARTPLGYM
GPDGVMGPMGLFLPNGHAGPHGQYGPAGKLIPKGK
HGPFGKRGPLGARGPTGFKMPKGLKGPMGVQGPQ
GHFVPFGLQGPTGQAGPRGGSQPAGLHGPLGAAGP
AGRCHPYGMVGPRGLAMPRGHQGPRGVYGPKGH (SEQ ID NO: 110) VPHVG (SEQ ID
NO: 36) LCST VHPGVG (SEQ ID NO: 37) LCST VPGAVG (SEQ ID NO: 38)
LCST VPGVAG (SEQ ID NO: 39) LCST APGVG (SEQ ID NO: 99) LCST VPGVA
(SEQ ID NO: 111) LCST VRPVG (SEQ ID NO: 40) LCST GRGDSPY (SEQ ID
NO: 41) UCST GRGDSPH (SEQ ID NO: 42) (for each) GRGDSPV (SEQ ID NO:
43) GRGDSPYG (SEQ ID NO: 44) UCST RPLGYDS (SEQ ID NO: 45) UCST and
RPAGYDS (SEQ ID NO: 46) LCST (for each) GRGDSYP (SEQ ID NO: 47)
UCST GRGDSPYQ (SEQ ID NO: 48) UCST GRGNSPYG (SEQ ID NO: 27) UCST
GRGDAPYQ (SEQ ID NO: 49) UCST (predicted) VPHSRNGG (SEQ ID NO: 51)
UCST VPHSRNGL (SEQ ID NO: 52) VPGHSHRDFQPVLHLVALNSPLSGGMRG LCST
(SEQ ID NO: 53) HTHQDFQPVLHLVALNTPLSGGMRGIRPGG LCST (SEQ ID NO:
54)
[0062] The polypeptides may include a combination of LCST and UCST
motifs which may convey both UCST and LCST transition behavior to
the polypeptide. The LCST and UCST motifs may be interspersed in
the polypeptide. For example, the LCST and USCT motifs may be
randomly distributed in the polypeptide, may alternate with each
other, may be consecutive, or may include spacer sequences between
them, or any possible combination thereof.
[0063] The polypeptides may include at least about 5, at least
about 10, at least about 11, at least about 12, at least about 13,
at least about 14, at least about 15, at least about 16, at least
about 17, at least about 18, at least about 19, at least about 20,
or at least about 25 of the motifs described herein and less than
about 1000, less than about 500, less than about 400, less than
about 300, less than about 275, less than about 250, less than
about 225, less than about 200, less than about 150, less than
about 100, less than about 75, less than about 50, or less than
about 30 of the motifs described herein. The polypeptides may
include only one repeated motif, or may include a number of
different motifs, as set forth above, which may or may not be
repeated. Polypeptides may be formed which are homopolymers of the
repeating units, or heteropolymers, including alternating
copolymers, periodic copolymers, block copolymers, and statistical
copolymers. Block polymers may include diblocks or triblocks of the
motifs described herein.
[0064] The motifs may be consecutive within the polypeptide, may be
separated by one or more spacer sequences, or a combination
thereof. The spacer sequences may include at least 1, at least 2,
least 3, at least 4, least 5, at least 6, least 7, at least 8,
least 9, at least 10, least 11, at least 12, least 13, at least 14,
least 15, at least 16, least 17, at least 18, least 19, at least
20, least 21, at least 22, least 23, at least 24, least 25, at
least 26, at least 27, at least 28, at least 29, at least 30, at
least 35, at least 40, at least 45, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 150, at
least 200, at least 250, or at least 300 amino acids.
[0065] Polypeptides may also be retro-polypeptides, including the
polypeptides or one or more of the motifs disclosed herein as a
reverse sequence reading from the C-terminus to the N-terminus,
rather than the N-terminus to the C-terminus.
[0066] The present disclosure facilitates the design of
biologically inspired polypeptides based on the identification and
reverse engineering of minima functional motifs found in natural
repetitive proteins, which confer a variety of structural or
functional properties. The present inventors discovered that amino
acid sequences seen to recur in natural repetitive proteins occur
as a subspace of particular sequences, which have been selected
through evolution, within a larger space of sequences displaying
similar properties.
[0067] The compositions disclosed herein can be used as materials
for a variety of biomedical and biotechnological applications,
including, without limitation, those pertaining to the uses of
elastin-derived biomaterials, silk-like biomaterials, resilin-like
biomaterials and elastin-like biomaterials (see, e.g., U.S. Pat.
No. 7,429,458; U.S. Pat. No. 6,852,834; U.S. Pat. No. 5,336,256;
U.S. Patent Application No. 2010/0015070; U.S. Pat. No. 7,674,882;
and U.S. Pat. No. 4,976,734, the disclosures of each of which are
herein incorporated by reference in their entireties).
[0068] Amino acid motifs and polypeptides disclosed herein may be
used in the design and synthesis of elastomeric-inspired
polypeptides, which may be engineered to confer or possess
environmental sensitivity and/or elasticity to protein-polymers,
while maintaining sequence diversity. A variety of useful
properties and applications are envisaged, including those
presented below.
[0069] The functional motifs described are sufficiently flexible to
be incorporated directly into bioactive polypeptides to confer
environmental sensitivity and elasticity to such polypeptides. For
example, the present disclosure makes it possible to transform a
protein bioactive peptide signal (e.g., cell adhesion peptides,
integrin inhibitors, anti-inflammation peptides, cell
differentiation, proliferation, angiogenic and anti-angiogentic
signals, etc.) into an environmentally responsive polypeptide
capable of acting as a "smart" drug that is environmentally
responsive.
[0070] Functional motifs may also be included in combination with
one or more other drugs or therapeutics transported as part of an
engineered self-assembled therapeutic delivery vehicle wherein the
vehicle itself may constitute an environmentally responsive
polypeptide based biodrug. The polypeptide may become bioactive
after undergoing a conformational change at the biological target
site. Functional motifs may also be included or introduced into a
scaffolding material wherein the functional motifs carry
biochemical cues for controlling cell-cell and cell-surface
interactions, inflammation, chemotaxis or any other relevant
biological process.
[0071] Environmentally responsive, bioactive polypeptides, may be
used as integrin inhibitors for anti-angiogenic therapy or as
active pro-angiogenic materials for tissue engineering
applications. The sequence diversity of the polypeptides described
herein may be exploited because different integrins recognize
alternative peptide sequences. For example, a4131 can recognize
EILDV (SEQ ID NO: 60) and REDV (SEQ ID NO: 61). Polypeptides
described herein having an unordered structure are suitable for
displaying these signals. Anti-inflammatory environmentally
responsive polypeptides may include one or more of a PG motif,
PX.sub.1G motif, or combination thereof. The motif may be an
anti-inflammatory peptide, such as FEWTPGWYQPY (SEQ ID NO: 59) and
modifications thereof, that prevent scaffold immunorejection.
[0072] The polypeptides described herein display similar elasticity
and environmental sensitivity to other elastin-like polypeptides
(ELPs), and may be utilized in applications which exploit the
properties of ELPs. The use of the motifs described herein allows
for control of the hydrophobicity profile of the polypeptides or
blocks within block-copolymer polypeptides, by substituting a
variety of residues in any of the positions available for
substitution within the functional motif. Polypeptides may thus be
designed, for example, to self-assemble into more complex and/or
defined structures, to gain control on the elastic force
(correlated with Pro content and hydrophobicity), or to improve the
environmental sensitivity of tags for protein purification.
[0073] Polypeptides described herein may also be used in drug and
therapeutic delivery applications for treating patients suffering
from a disease or condition. Such vehicle delivery applications may
include the formation of nanoparticles from environmentally
responsive polypeptides and their delivery to a biological site
such as an organ, tissue, tumor, wound site or disease site. The
tumor, wound or disease site may be localized or systemic in a
patient. Targeted delivery may be achieved by recognition, binding
or affinity of a particular receptor or other molecule that is
associated with the tumor, wound or disease by the nanoparticle.
The nanoparticles may self-assemble around a therapeutic. The
therapeutic may be hydrophobic or hydrophilic. For example, drug
and therapeutic delivery vehicles comprising polypeptides described
herein that respond to a pH stimulus or temperature change at the
microenvironment of the biological site, may be triggered to
release a chemotherapeutic cargo in the biological site. The
ability to fine tune the pH responsiveness of drug and therapeutic
delivery vehicles may be achieved by selecting a polypeptide having
amino acid residues in the motifs which have appropriate pKa
values, such that they change their ionization state at relevant pH
values.
[0074] Polypeptides described herein may be bioactive, or may lose
bioactivity or become bioactive after delivery to a biological
site. Bioactive refers to the ability to have biological activity
at a biological site, and may include the ability to induce
biological effects, therapeutic activity, or a combination
thereof.
[0075] Fusion proteins may be formed which include an
environmentally responsive polypeptide operably connected to a
polypeptide of interest. Fusion proteins may be generated by
generating a polynucleotide which includes a polynucleotide
encoding an environmentally responsive polypeptide operably
connected to a polynucleotide encoding a polypeptide of interest
and expressing a polypeptide from the polynucleotides. The
expressed polypeptide contains the environmentally responsive
polypeptide connected or fused to the polypeptide of interest.
Optionally a linker sequence may be included between the
polynucleotides and fused polypeptides. The linker sequence may be
at least 1, least 2, least 3, least 4, least 5, least 6, least 7,
least 8, least 9, least 10, least 15 amino acids and less than 200,
150, 100, 75, 50, 40, 30 or 20 amino acids. Expression may be
carried out, for example in a bacterial, yeast or mammalian cell,
with the appropriate promoter sequence. Fusion proteins may also be
generated by chemical synthesis, or by chemically attaching
naturally or chemically synthesized peptides and polypeptides
together.
[0076] The environmentally sensitive polypeptide may also be
chemically conjugated to molecules such as therapeutics,
carbohydrates, synthetic polymers, polynucleotides and
oligonucleotides, including DNA, RNA, as well chemically
synthesized small molecules.
[0077] The present disclosure is not limited in its application to
the details of construction and the arrangement of components set
forth in the following description or illustrated in the drawings.
The invention described in the present disclosure is capable of
other embodiments and of being practiced or of being carried out in
various ways. Also, the phraseology and terminology used herein is
for the purpose of description and should not be regarded as
limiting.
[0078] The use of "including," "comprising," "having,"
"containing," "involving," and variations thereof herein, is meant
to encompass the items listed thereafter and equivalents thereof as
well as additional items. For the purposes of promoting an
understanding of the principles of the present disclosure,
reference will now be made to preferred embodiments and specific
language will be used to describe the same.
[0079] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. at least one) of the grammatical object of
the article. By way of example, "an element" means at least one
element and can include more than one element.
[0080] As used herein, the term "about," when referring to a value
or to an amount of mass, weight, time, volume, concentration or
percentage is meant to encompass variations of in some
embodiments.+-.20%, in some embodiments.+-.10%, in some
embodiments.+-.5%, in some embodiments.+-.1%, and in some
embodiments.+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed methods.
EXAMPLES
[0081] Materials and Methods
[0082] Gene synthesis of EIPs: EIPs can be synthesized by standard
molecular biology techniques suitable for the synthesis of genes
encoding repetitive protein-polymers, some of which have been
previously described (see, e.g., McDaniel, J. R. et al. (2010)
Biomacromolecules 10.1021/bm901387t; Meyer, D. E. et al. (2002)
Biomacromolecules 3:357-367). Many of the EIP genes herein reported
were synthesized by a novel method recently developed by the
inventors, which are described elsewhere herein. Regardless of the
gene synthesis method used for their construction, the DNA sequence
of all of the genes encoding EIP protein-polymers characterized
herein were verified by direct DNA sequencing (Eton Bioscience
Inc., NC, USA). Before expression, an N-terminal leader sequence
encoding for Met-Ser-Lys-Gly-Pro (SEQ ID NO: 62) and a C-terminal
His-tag tail encoding for His-His-His-His-His-His-Y (SEQ ID NO: 63)
were incorporated into the genes, unless indicated. The properties
of the compositions subject to the present disclosure are
independent of the aforementioned leader and trailer sequences;
however, the incorporation of a His-tag sequence typically results
in a significant increase in the transition temperature of
polypeptides, particularly those of low molecular weight (e.g.,
<20 KDa).
[0083] Expression and Characterization of EIPs: Before large-scale
expression, starter cultures (3-5 mL) of TB media supplemented with
100 .mu.g/mL ampicillin were inoculated with transformed cells from
DMSO stocks stored at -80.degree. C., and incubated overnight at
37.degree. C. while shaking at 250 rpm. The starter cultures were
then centrifuged at 3000 g for 2 min, and resuspended in 1 mL of
fresh TB medium. Expression cultures (4 L flasks containing 1 L of
TB media with 100 .mu.g/mL ampicillin) were inoculated with the
resuspended starter culture and incubated at 37.degree. C. with
shaking at 200 rpm. After 6-7 h of growth, expression was induced
by the addition of IPTG to a final concentration of 1 mM. The cells
were harvested 24 h after inoculation, and purified by inverse
transition cycling (ITC) as previously described, or, in a limited
number of cases, by His-tag purification following the instructions
of the manufacturer (Pierce, USA) (see, e.g., Christensen, T. et
al. (2009) Protein Science 18:1377-1387). To characterize the
inverse transition temperature of EIPs, the optical density of EIP
solutions (25-50 .mu.M), prepared in PBS or PBS supplemented with
NaCl and/or 8M Urea as indicated, was monitored at a wavelength of
350 nm (0D.sub.350) as a function of temperature, at a heating rate
of 1.degree. C. min.sup.-1, on a Cary 300 UV-visible
spectrophotometer equipped with a multicell thermoelectric
temperature controller (Varian Instruments, Walnut Creek, Calif.).
The derivative of the optical density with respect to temperature
was numerically calculated, and the Tt was defined as the
temperature at the maximum of the turbidity gradient.
[0084] Bioinformatics Studies: The amino acid sequence of different
elastomeric and non-elastomeric proteins was retrieved as FASTA
(.txt) filed from the National Center for Biotechnology Information
protein database. In-house custom methods were implemented in
MATLAB software (MathWorks, Natick, Mass.) to map the location of
elastin-like polypeptide motifs (e.g., VPGXG (SEQ ID NO: 3), IPGXG
(SEQ ID NO: 5)) among the sequence of these proteins, the
occurrence of generalized P(X.sub.n)G motifs (where n indicated the
number of X residues separating a given Proline and Glycine
residue, and varies as 1.ltoreq.n.ltoreq.5), the hydrophobicity
profiles of amino acids associated with residues participating or
surrounding these motifs, the distance between them, and the
precise location of Glycine residues in relation to Glycine
residues participating in P(X.sub.n)G motifs. Two different
hydrophobicity scales were considered in these analyses, the scale
proposed by Kyte and Doolittle and Urry et al. (see, e.g., Kyte, J.
et al. (1982) J. Mol. Biol. 157:105-132; Urry D. W. (1992)
supra).
TABLE-US-00002 TABLE 1 Sequence information of the different
polypeptides used in the bioinformatics studies. Accession Protein
Species Number (GI) Elastin Homo sapiens 182021 Elastin Bos taurus
28461173 Elastin Mus musculus 31542606 Elastin Rattus norvegicus
55715827 Elastin Macaca mulatta 13182892 Elastin b Danio rerio
(Zebrafish) 114326248 Elastin a Danio rerio (Zebrafish) 121583675
Alpha-1 Collagen Type I Homo sapiens 553615 Collagen Type III alpha
1 Homo sapiens 4502951 Collagen Type II alpha 1 Homo sapiens
111118974 Collagen Type X alpha 1 Homo sapiens 120659966 Collagen
Type VIII alpha 2 Homo sapiens 32964830 Fibrillin 1 Homo sapiens
46559358 Dragline silk fibroin Nephila clavipes 159714 (Spidroin 2)
flagelliform silk protein Nephila clavipes 2833649 Fibulin 5
precursor Homo sapiens 19743803 Fibrillin Homo sapiens 1335064
Resilin Isoform B Drosophila melanogaster 45552671 Resilin Isoform
A Drosophila melanogaster 7302880 High molecular weight Elymus
alashanicus 84181091 gluten subunit Titin Homo sapiens 1212992
Fibroin-3 (ADF-3) Araneus Diadematus 1263287 Protein PRQFV-amide
Aplysia californica 74842069 FMRFamide-related Lymnaea stagnalis
1169643 neuropeptides Transcription elongation Schizosaccharomyces
pombe 74581925 factor spt5
[0085] Gene synthesis: The phase transition biopolymers described
in the present invention were synthesized by standard molecular
biology techniques suitable for the synthesis and expression of
genes encoding repetitive protein-polymers. The genes encoding for
the biopolymers herein reported were synthesized by three different
methods, described below.
[0086] 1. All genes, unless indicated, were synthesized using
OERCA. Briefly, single stranded DNA sequences were designed
encoding for 1 to 5 copies of the amino acid motif of
interest--depending on the motif length--, which were then
circularized using a ligase. The circular DNA was amplified and
extended using primers specific for the 5' and 3' ends of the
linear DNA using a polymerase with strand displacement activity in
a PCR-type reaction that resulted in gene polymerization by two
means: first, by way of rolling circle amplification, and second,
by overlap elongation of the extended genes that had rolled from
the circle. This resulted in a library containing oligomers with
various numbers of repeats of the starting monomer unit (that
already included 1-5 copies of the motif of interest), which were
blunt ligated into a modified pET25 vector and transformed into
BL21 cells. Clones having genes of various sizes and that were
inserted in the correct orientation were screened, by way of colony
PCR and direct DNA sequencing (Eton Bioscience Inc., NC, USA). DMSO
stocks of all clones that harbored one gene that encoded for any
number of repeats of the motif of interest were prepared.
[0087] 2. Genes encoding for biopolymers with repeat units longer
than 50 amino acids and with randomized composition were purchased
from Mr. Gene and cloned into a modified pET24 vector for
expression in E. coli. We prepared DMSO stocks of the clones that
harbored the gene of interest.
[0088] 3. Genes encoding for biopolymers with the following motifs
were synthesized by Pre-RDL as recently described by the inventors
[McDaniel et al. 2010]: GRGDSPYQ (SEQ ID NO: 48), GRGNSPYG (SEQ ID
NO: 49), LGAPVG (SEQ ID NO: 25),
AVPGVGAVPGVGAVPGVGAVPGVGAVPGVGCVPGVG (SEQ ID NO: 64), and
VPAGVGVPAGVGVPAGVGVPAGVGVPAGVGVPCGVG (SEQ ID NO: 65). Briefly, we
designed oligonucleotides for the sense and antisense strands of
genes encoding for 1-5 copies of the motifs of interest, which
annealed leaving a 3' GG overhang on the sense strand and a 3' CC
overhang on the antisense stand to allow for concatemerization. The
concatemers were ligated into a modified pET24 vector and
transformed into E. coli, and the resulting colonies were screened
by colony PCR and direct DNA sequencing (Eton Bioscience Inc., NC,
USA). We prepared DMSO stocks of all clones that harbored one gene
that encoded for any number of repeats of the motif of
interest.
[0089] Before expression, an N-terminal leader sequence encoding
for Met-Ser-Lys-Gly-Pro (SEQ ID NO: 62) and a C-terminal His-tag
tail encoding for His-His-His-His-His-His-Y (SEQ ID NO: 63) were
incorporated into the genes, except for the genes synthesized by
(2) and (3), which lacked the His-tag sequence. The properties of
the compositions subject of the present invention are independent
of the aforementioned leader and trailer sequences; however, the
incorporation of a His-tag sequence typically results in a
significant increase in the transition temperature of polypeptides,
particularly those of low molecular weight (e.g., <20 KDa).
[0090] Expression and characterization of EIPs. Before large-scale
expression, starter cultures (3-5 mL) of TB media supplemented with
100 .mu.g/mL ampicillin were inoculated with transformed cells from
DMSO stocks stored at -80.degree. C., and incubated overnight at
37.degree. C. while shaking at 250 rpm. The starter cultures were
then centrifuged at 3000 g for 2 min, and resuspended in 1 mL of
fresh TB medium. Expression cultures (4 L flasks containing 1 L of
TB media with 100 .mu.g/mL ampicillin) were inoculated with the
resuspended starter culture and incubated at 37.degree. C. with
shaking at 200 rpm. After 6-7 h of growth, expression was induced
by the addition of IPTG to a final concentration of 1 mM. The cells
were harvested 24 h after inoculation, and purified by inverse
transition cycling (ITC) as previously described (Christensen et
al. 2009). To characterize the inverse transition temperature of
phase transition biopolymers, the optical density of biopolymer
solutions (25-50 .mu.M), prepared in PBS or PBS supplemented with
NaCl and/or 1-8M Urea as indicated, was monitored at a wavelength
of 350 nm (0D350) as a function of temperature, at a heating rate
of 1.degree. C. min-1, on a Cary 300 UV-visible spectrophotometer
equipped with a multicell thermoelectric temperature controller
(Varian Instruments, Walnut Creek, Calif.). The derivative of the
optical density with respect to temperature was numerically
calculated, and the Tt was defined as the temperature at the
maximum of the turbidity gradient.
[0091] Bioinformatics studies. The amino acid sequence of different
Pro- and Gly-rich proteins was retrieved as FASTA (.txt) files from
the National Center for Biotechnology Information protein database.
In-house custom methods were implemented in MATLAB software
(MathWorks, Natick, Mass.) to map the location of elastin-like
polypeptide motifs (e.g., VPGXG (SEQ ID NO: 3), IPGXG (SEQ ID NO:
5)) among the sequence of these proteins, the occurrence of
generalized P(X.sub.n)G motifs (where n indicates the number of X
resides separating a given Proline and Glycine reside, and varies
as 1.ltoreq.n.ltoreq.5), the hydrophobicity profiles of amino acids
associated with residues participating or surrounding these motifs,
the distance between them, and the precise location of Glycine
residues in relation to Glycine residues participating in
P(X.sub.n)G motifs. Two different hydrophobicity scales were
considered in these analyses, the scale proposed by Kyte and
Doolittle (1982) and the scale proposed by Urry et al. 1992.
[0092] Results
[0093] Simple bioinformatics methods were developed and implemented
to visualize and quantify minima functional motifs in elastomeric
proteins. FIGS. 1A-1C show a digital map of the distribution and
conservation of the canonical ELP motif VPGXG (SEQ ID NO: 3) along
the sequence of elastin from various species. Relatively poor
sequence conservation and low sequence coverage were observed. A
minima functional P(X.sub.n)G motif was mapped, which encompasses
multiple potential arrangement of Proline and Glycine residues,
among elastin sequences. The results showed a surprisingly high
degree of sequence conservation and sequence coverage, mostly
corresponding to PG units (i.e., n=0) (FIG. 1C). A similar map was
drawn from other elastomeric and non-elastomeric proteins where
Proline and Glycine residues occur with high frequency. The
occurrence of P(X.sub.n)G motifs other than the one observed in
elastin sequences, particularly those where n=1 and n=4, were
observed (see FIGS. 2A-2D). Moreover, the distribution of these
residues was random, as contrasting maps were observed for
P(X.sub.n)G and G(X.sub.n)P motifs (see FIGS. 2A-2D). These maps
also uncovered a potential role for Gly residues closely positioned
surrounding the P(X.sub.n)G motif, as observed when comparing the
elastomeric flagelliform silk (FIG. 2A), where Gly usually occurs
one residue before Proline, with elastin, gluten or resilin (FIGS.
2B-2D), where Gly occurs two or more positions before Proline.
There was also evidence of a high abundance of a PX.sub.4G motif in
resilin, but no evidence for PX.sub.2G and PX.sub.3G motifs was
observed (see FIGS. 3A-3E). Only silk proteins had a large
percentage of GPX.sub.n motifs.
[0094] Elastomeric proteins display different properties and
several mechanisms may be responsible for such differences; one
such mechanism may relate to protein hydrophobicity. Elastomeric
proteins, particularly elastin, may include an association of
hydrophobic and hydrophilic (cross-linking) domains. A more
detailed profiling of the hydrophobicity of key residues
participating in the identified PX.sub.nG motifs was conducted. The
hydrophobicity of these residues among elastin proteins, showed a
distribution of hydropathy indices, which remained largely
hydrophobic for bovine and Homo sapiens, but extended into more
hydrophilic values for elastin proteins from Zebrafish and Mus
musculus (see FIG. 4). Different evolutionary constraints
experienced by these species may have resulted in the selection of
amino acids covering various regions of a space of sequences all
ascribing to a general PX.sub.nG motif. Evolutionary bias toward
the localization of Gly residues two positions before the Pro (Pm2;
Proline minus 2 residues) and one (Gp1; Gly plus 2 residues) or two
(Gp2; Gly plus 2 residues) positions after the Gly in the PX.sub.nG
motif was observed, along with a very low frequency of Gly residues
occurring one position before the Pro (Pm1; Proline minus 1
residue). The abundant PX.sub.1G motif in collagen (FIG. 3B),
however, occurs almost entirely with Gly at Pm1, with no other
position surrounding the motif being biased toward any particular
residue or hydropathy (see FIG. 5). Similar analyses were performed
on a larger pool of elastomeric proteins and amino acids covering a
broader range of hydropathy indices were explored; a similar bias
in the distribution of Gly residues was found. For instance,
resilin and gluten PX.sub.0G motifs are surrounded primarily by
hydrophilic residues, such that the selection of hydrophobic amino
acids in these positions may not be a prerequisite for the elastic
behavior of these proteins. Similarly, the overall hydropathy of
Dragline silk and Flagelliform silk are almost identical, being
-0.40 and 0.37 (Kyte-Doolittle scale), respectively. The
hydrophobicity of the residues surrounding the abundant PX.sub.0G
motif in Dragline silk and Flagelliform silk (i.e., Pm2, Pm1, Gp1
and Gp2 in FIG. 5) show an average hydropathy for the neighboring
residues of -0.16 and -1.2, respectively. Resilin shows an abundant
(FIG. 3B) highly conserved PX.sub.4G motif populated by hydrophilic
residues (FIG. 7); the conserved PX.sub.4G motif is a continuous
motif not abundant in related elastomeric proteins (FIG. 3 A-B;
FIG. 7).
[0095] The results revealed that distribution of Pro and Gly
residues may be responsible for the elasticity and/or environmental
responsiveness displayed by these proteins. Collagen, although not
elastomeric, is thermoresponsive and presents a large number of
both PX.sub.0G and PX.sub.1G motifs, and has a much larger Pro
content than the proteins analyzed herein, particularly Pro
residues at Pm1 and Pm2 at PX.sub.0G and PX.sub.1G motifs,
respectively. The PX.sub.1G motifs have relatively low abundance in
most elastomeric proteins described.
[0096] The canonical elastin-like motif includes PX.sub.0G, where
only the X residue in the pentapeptide motif VPGXG (SEQ ID NO: 3)
was previously believed to be able to accept amino acids of any
hydrophobicity. However, bioinformatic studies showed the
occurrence of PX.sub.0G motifs with at least 4 positions
surrounding the PG dipeptide covering a broad spectrum of
hydropathy indices; indeed, there was no evidence for larger
sequence diversity at Gp1 (equivalent to X in VPGXG; SEQ ID NO: 3)
among elastin sequences and other elastomeric proteins (FIGS. 4 and
5). Therefore, to further test the hypothesis, a large data set of
polypeptides were created incorporating PG, PX.sub.1G,
PX.sub.1X.sub.2G (SEQ ID NO: 16) and PX.sub.1X.sub.2X.sub.3X.sub.4G
(SEQ ID NO: 66) motifs while varying the residues that surround or
constitute the P(X.sub.n)G motifs, and engineering Gly residues at
different positions around the P(X.sub.n)G motif to study the role
of Gly at these positions, as suggested by the bioinformatics study
below in Table 2.
TABLE-US-00003 TABLE 2 Minima functional environmentally sensitive
motifs that were reverse engineered from the sequence of a variety
of elastomeric and non-elastomeric proteins (see Table 2), and the
experimental evidence from corresponding EIP motifs demonstrated to
display elasticity and/or environmental sensitivity. EIPs of
various lengths for a single repeat unit were generated to study
the role of molecular weight on their behavior. Equivalent EIP
constructs Gp2, Minimum functional Pm2, motif Repeat unit* Z and X
values Pm1 P(X.sub.0)G AVPGVG Z.sub.1 = A, Z.sub.2 = V, Z.sub.3 =
V, Z.sub.4 = G Gp2 = G Z.sub.1Z.sub.2PGZ.sub.3Z.sub.4 (SEQ ID NO:
8) (SEQ ID NO: 9) VAPGVG Z.sub.1 = V, Z.sub.2 = A, Z.sub.3 = V,
Z.sub.4 = G Gp2 = G (SEQ ID NO: 67) GVPGAV Z.sub.1 = G, Z.sub.2 =
V, Z.sub.3 = A, Z.sub.4 = V Pm2 = G (SEQ ID NO: 68) GVPGVA Z.sub.1
= G, Z.sub.2 = V, Z.sub.3 = V, Z.sub.4 = A Pm2 = G (SEQ ID NO: 69)
TVPGVG Z.sub.1 = T, Z.sub.2 = V, Z.sub.3 = V, Z.sub.4 = G Gp2 = G
(SEQ ID NO: 70) TVPGAG Z.sub.1 = T, Z.sub.2 = V, Z.sub.3 = A,
Z.sub.4 = G Gp2 = G (SEQ ID NO: 71) GAPGVG Z.sub.1 = G, Z.sub.2 =
A, Z.sub.3 = V, Z.sub.4 = G Gp2 = G (SEQ ID NO: 72) & Pm2 = G
AVPGVA Z.sub.1 = A, Z.sub.2 = V, Z.sub.3 = V, Z.sub.4 = A Gp2
<> G (SEQ ID NO: 73) & Pm2 <> G GAPGGG Z.sub.1 = G,
Z.sub.2 = A, Z.sub.3 = G, Z.sub.4 = G Gp2 = G (SEQ ID NO: 74) &
Pm2 = G GAPGAG Z.sub.1 = G, Z.sub.2 = A, Z.sub.3 = A, Z.sub.4 = G
Gp2 = G (SEQ ID NO: 75) & Pm2 = G GGPGAG Z.sub.1 = G, Z.sub.2 =
G, Z.sub.3 = A, Z.sub.4 = G Gp2 = G (SEQ ID NO: 76) & Pm2 = G
& Pm1 = G P(X.sub.1)G GVPAGVG Z.sub.1 = G, Z.sub.2 = V, X = A,
Z.sub.3 = V, Gp2 = G Z.sub.1Z.sub.2PX.sub.1GZ.sub.3Z.sub.4 (SEQ ID
NO: 77) Z.sub.4 = G & (SEQ ID NO: 13) Pm2 = G VGPVGVG Z.sub.1 =
V, Z.sub.2 = G, X = V, Z.sub.3 = V, Gp2 = G (SEQ ID NO: 78) Z.sub.4
= G & Pm1 = G GVPTGVG Z.sub.1 = G, Z.sub.2 = V, X = T, Z.sub.3
= V, Gp2 = G (SEQ ID NO: 79) Z.sub.4 = G & Pm2 = G GAPVGVG
Z.sub.1 = G, Z.sub.2 = A, X = V, Z.sub.3 = V, Gp2 = G (SEQ ID NO:
80) Z.sub.4 = G & Pm2 = G VAPVGVA Z.sub.1 = V, Z.sub.2 = A, X =
V, Z.sub.3 = V, Gp2 <> G (SEQ ID NO: 81) Z.sub.4 = A &
Pm2 = G GAPFGFA Z.sub.1 = G, Z.sub.2 = A, X = F, Z.sub.3 = F, Pm2 =
G.sup..PSI. (SEQ ID NO: 82) Z.sub.4 = A AIPMGAG Z.sub.1 = A,
Z.sub.2 = I, X = M, Z.sub.3 = A, Gp2 = G.sup..PSI. (SEQ ID NO: 83)
Z.sub.4 = G GFPTGGL Z.sub.1 = G, Z.sub.2 = F, X = T, Z.sub.3 = G,
Pm2 = G.sup..PSI. (SEQ ID NO: 84) Z.sub.4 = L LAPFGMG Z.sub.1 = L,
Z.sub.2 = A, X = F, Z.sub.3 = M, Gp2 = G.sup..PSI. (SEQ ID NO: 85)
Z.sub.4 = G GLPAGMG Z.sub.1 = G, Z.sub.2 = L, X = A, Z.sub.3 = M,
Gp2 = G (SEQ ID NO: 86) Z.sub.4 = G & Pm2 = G.sup..PSI.
P(X.sub.2)G GVPAVGV Z.sub.1 = G, Z.sub.2 = V, X.sub.1 = A, X.sub.2
= V, Pm2 = G Z.sub.1Z.sub.2PX.sub.1X.sub.2GZ.sub.3 (SEQ ID NO: 87)
Z.sub.3 = V (SEQ ID NO: 134) GVPHVGV Z.sub.1 = G, Z.sub.2 = V,
X.sub.1 = H, X.sub.2 = V, Pm2 = G (SEQ ID NO: 88) Z.sub.3 = V
VGPAVGV Z.sub.1 = V, Z.sub.2 = G, X.sub.1 = A, X.sub.2 = V, Gp2 = G
(SEQ ID NO: 89) Z.sub.3 = V & Pm1 = G VTPAVGV Z.sub.1 = V,
Z.sub.2 = T, X.sub.1 = A, X.sub.2 = V, Gp2 <> G (SEQ ID NO:
90) Z.sub.3 = V & Pm2 <> G P(X.sub.4)G GVPSALYGVG Z.sub.1
= G, Z.sub.2 = V, X.sub.1 = S, X.sub.2 = A, Gp2 = G
Z.sub.1Z.sub.2PX.sub.1X.sub.2X.sub.3X.sub.4GZ.sub.3Z.sub.4 (SEQ ID
NO: 91) X.sub.3 = L, X.sub.4 = Y, Z.sub.3 = V, Z.sub.4 = G &
(SEQ ID NO: 15) Pm2 = G GVPSDDYGQG Z.sub.1 = G, Z.sub.2 = V,
X.sub.1 = S, X.sub.2 = D, Gp2 = G (SEQ ID NO: 92) X.sub.3 = D,
X.sub.4 = Y, Z.sub.3 = Q, Z.sub.4 = G & Pm2 = G GVPSDDYGVG Gp2
= G (SEQ ID NO: 93) Z.sub.1 = G, Z.sub.2 = V, X.sub.1 = S, X.sub.2
= D, & X.sub.3 = D, X.sub.4 = Y, Z.sub.3 = V, Z.sub.4 = G Pm2 =
G *Letters underlined are not part of the EIP repeat unit in the
constructed polypeptides, but correspond to a residue in the n .+-.
1 repeat unit as it is presented in tandem. .sup..PSI.The
functionality of these motifs was assessed as part of a single
polypeptide generated by using a method that randomized the
selection of amino acids for 5 hexapeptides following a
Z.sub.1Z.sub.2PX.sub.1GZ.sub.3 (SEQ ID NO: 22) motif, which were
then repeated in tandem. The design principle entailed having a G
residue at either Pm2 or Gp2. A normal distribution was used for
selection of residues with a target Hi of 2 for both X and Z
residues and a standard deviation of 1.5. The sequence of such
randomized polypeptide is: (GAPFGFAIPMGAGFPTGGLAPFGMGLPAGM).sub.n
((SEQ ID NO: 12).sub.n). **These elastic sequences display thermal
stability and solubility due to the large contribution of the
hydrophilic aspartate residues confined within the PXXXXG (SEQ ID
NO: 94) motif to the inverse transition temperature of these
sequences.
[0097] The environmental responsiveness of the motifs in Table 2
(see FIGS. 8-16) was characterized and demonstrated the robustness
of the P(X.sub.n)G motifs to confer environmental sensitivity and
elasticity to polypeptides, and the possibility to engineer
flexible functional Z.sub.mP(X.sub.n)GZ.sub.k (SEQ ID NO: 95)
motifs, wherein Z.sub.m and Z.sub.k are each amino acids of any
hydrophobicity which surround the P(X.sub.n)G functional unit and
n, m, k.ltoreq.4. For simplicity and clarity, the data in FIG. 8
through 16 is presented by grouping all the polypeptides described
by a common Z.sub.mP(X.sub.n)GZ.sub.k (SEQ ID NO: 95) motif
[0098] The characterization of retro-EIPs, in which the sequence of
the motif was backbone reversed (see Table 3 below) while
regenerating a PXnG motif typically with different n value, further
demonstrated the robustness of these motifs since environmentally
sensitivity was maintained in all cases (see FIG. 17). Backbone
reversal of the motif APGVG (Table 2) would result in an EIP with a
PX1X2X3G (SEQ ID NO: 96) motif, which constitutes an additional
minima functional motif ZmP(Xn)GZk (SEQ ID NO: 95) where n=3.
TABLE-US-00004 TABLE 3 Backbone-reversed-retro-EIPs. Modified Motif
Retro-motif retro-motif* Minimum Minimum Minimum unit EIP unit EIP
unit EIP PX.sub.0G VPGVG PX.sub.1G VGPVG PX.sub.1G VAPVG (SEQ ID
(SEQ ID (SEQ ID NO: 97) NO: 98) NO: 27) PX.sub.0G VAPGVG PX.sub.2G
VTPAVG PX.sub.2G VGPAVG (SEQ ID (SEQ ID (SEQ ID NO: 67) NO: 17) NO:
18) PX.sub.1G VPAGVG PX.sub.1G VGAPVG -- (SEQ ID (SEQ ID NO: 33)
NO: 24) IDX.sub.2G VPAVG PX.sub.1G VAPVG -- (SEQ ID (SEQ ID NO: 6)
NO: 27) *The modified (substituted or inserted) residue from the
original retro-motif is shown underlined. The sequence of an EIP
motif as read from the N- to C-terminus was reversed so that the
sequence was identical when read from the C- to N- terminus. In
addition, whenever a Gly residue occurred at Pm1 as a result of
backbone reversal, a modified retro-motif was synthesized in which
an Ala or Thr residue was substituted for Gly, in order to study
the effect of Gly at Pm1.
[0099] The data showed that biased selection of amino acids in key
motifs observed in repetitive proteins is representative of the
evolutionary constraints experienced by different species. A
surprising bias in the localization of Gly residues at Pm2 and Gp2
(at either position or simultaneously occurring for a given
PX.sub.nG repeat unit) surrounding PX.sub.nG motifs in elastomeric
proteins (see FIGS. 4-6) is not a prerequisite for the reversible
phase transition behavior displayed by elastomeric-inspired
polypeptides, as demonstrated by EIPs with motifs VAPVG (SEQ ID NO:
27) (FIG. 13) and APGVG (SEQ ID NO: 99) (FIG. 11). This observation
reinforces the possibility of constructing truly general motifs as
described in Table 2, where Z residues do not have to be restricted
to Gly at either Pm1 or GP2, while still preserving the
environmental sensitivity and elasticity observed in other
Z.sub.mP(X.sub.1)GZ.sub.k (SEQ ID NO: 95) and Z.sub.mPGZ.sub.k (SEQ
ID NO: 100) motifs. In addition, the demonstration of the
environmental sensitivity of a randomized EIP with repeat unit Z
.sub.1Z.sub.2PX.sub.1GZ.sub.2Z.sub.3Z.sub.4PX.sub.2GZ.sub.6Z.sub.7Z.sub.8-
PX.sub.3GZ.sub.9Z .sub.10Z
.sub.11PX.sub.4GZ.sub.12Z.sub.13Z.sub.14PX.sub.5GZ (SEQ ID NO: 101)
displaying a minima functional PX.sub.1G motif constitutes a step
further in the design of "smart" protein-polymers (see FIG.
12A).
[0100] Regarding the role of neighboring Gly residues in the
functionality of the P(Xn)G motif, FIG. 18 demonstrates that Gly at
Pm1 of a P(Xn)G facilitates self-assembly, presumably by making the
packing of the polypeptide chains more efficient due to reduced
steric hindrance provided by the side-chain motifs having a more
hydrophobic residue at Pm1, and by promoting the formation of
stable non-reversible fibrillar structures rather than driving the
self-assembly of fractal structures. Sheparavych et al. have shown
that for other self-assembling peptides, the disruption of the
peptide secondary structure, particularly their helical
conformation, is key to the self-assembly process in this fractal
manner (Sheparavych, R. et al. (2009) Biomacromolecules
10:1955-1961). Interestingly, the bioinformatics analyses only
pointed to the occurrence of Gly at Pm1 for collagen and silks, and
in both cases, abundant secondary and tertiary structures are
present, which is in agreement with the finding that Gly at Pm1
increases the propensity of these motifs to drive irreversible
phase separation and disruption of elasticity above a critical
temperature. In addition, it has also been shown that other
interesting self-assembly properties for PX1X2G (SEQ ID NO: 102)
motifs, as shown in FIG. 19, and as reported in the literature for
the ELP with sequence VPAVG (SEQ ID NO: 6), which also carries a
PX1X2G (SEQ ID NO: 102) motif (see, e.g, Bessa, P. C. et al. (2009)
supra).
[0101] An additional feature of the EIP motifs disclosed in the
present disclosure is the possibility to exploit their
environmental sensitivity for their efficient purification, in an
analogous manner to the purification schemes in use for the
preparation of ELPs. It has been observed by the inventors that
even those EIPs that display heat-irreversible phase separation,
typically display reversible phase separation in response to
changes in buffer ionic strength, and this property has been
exploited for their high yield purification. However, the
expression (in E. coli) and purification of EIPs with Gly at Pm1 is
somewhat more difficult compared with the ease of purification of
other EIPS, primarily due to their tendency to form insoluble
aggregates and form inclusion bodies during protein expression.
Interestingly, although Gly at Pm1 may drive the heat-irreversible
phase separation, it does not necessarily disrupt the reversible
phase separation when using an orthoganol stimulus, such as buffer
ionic strength.
[0102] It was reasoned that EIPs displaying heat-irreversible
inverse phase transition can be engineered as elastomeric or
non-elastomeric materials if cross-linked below or above the
threshold temperature for the heat-irreversible phase separation of
a given EIP. This is in accordance with Urry's observation that
potential elastomers display reversible aggregation, whereas
non-elastomeric polypeptides display irreversible aggregations
(U.S. Pat. No. 5,250,516). Noteworthy, the cross-linking conditions
could be readily adjusted to tune the transition temperature (Tt)
of the polypeptide to a temperature range below the threshold
temperature, by exploiting the sensitivity of EIPs to changes in
buffer ionic strength. In addition, it has also been observed by
the inventors that such threshold temperature is typically above
body temperature, so that devices composed of EIPs displaying
heat-irreversible phase separation should not be compromised upon
implantation, especially if cross-linked. In addition,
heat-irreversible phase separation or non-elastomeric behavior may
be favored by engineering Gly residues at Pm1 or PX.sub.nG minima
functional motifs that would promote the formation of stable
perhaps crystallizable structures above a threshold temperature
above the Tt. A recent study by Chen and Guan supports the idea
that the localization of Pro and Gly residues in the canonical ELP
motif VPGXG (SEQ ID NO: 3) serves a role in disrupting secondary
structures that would otherwise prevent the highly elastic behavior
and dynamic nature of these polypeptides, and rather promote the
formation of random coils or dynamic low stability conformations as
those based on .beta.-turns and Polyproline-II conformations (Chen,
Y. et al. (2009) J. Am. Chem. Soc. DOI:1021/ja9104446). Therefore,
the ability to engineer the development of such ordered structures
in unordered EIPs may provide additional means to control the
elasticity of a protein-polymer in a stimuli-controlled manner.
[0103] In addition, the present disclosure describes the thermally
responsive behavior of resilin-inspired polypeptides. Resilin-like
polypeptides with the 11 residue repeat AQTPSSQYGAP (SEQ ID NO:
103) have been regarded in the literature as unordered polypeptides
and were not reported to show thermally responsive behavior. A
highly conserved YGAP (SEQ ID NO: 104) motif was suspected to be
required for the properties, namely elasticity and resilience, of
this polypeptide (see, e.g., Nairn, K. M. et al. (2008) supra). In
contrast, the present disclosure indentifies a functional PX.sub.4G
motif in resilin, which can also be identified in the repeat unit
AQTPSSQYGAP (SEQ ID NO: 103), and demonstrated its environmental
sensitivity (FIG. 16) and the possibility to generalize the
sequence.
[0104] The present disclosure demonstrates the possibility to
synthesize bioactive EIPs for tissue engineering and regenerative
medicine applications by synthesizing EIPs incorporating the
bioactive potent proangiogenic GXXPG (SEQ ID NO: 21) motif found in
elastin (see, e.g., Robinet, A. et al. (2005) J. Cell Science
118:343-356), which display identical behavior to nonbioactive
conventional elastin-like polypeptides and thus display remarkable
environmental sensitivity and elasticity (FIG. 20). This is the
first demonstration of an elastic, environmentally sensitive
polypeptide carrying a potent chemokine, since non-elasticity of
the VAPGVG (SEQ ID NO: 67) hexapeptide has frustrated major
engineering efforts exploiting the functionality of this bioactive
motif.
[0105] A large number of genetically encoded protein-based polymers
were synthesized that span the entire range of Pro-X.sub.n-Gly
arrangements observed in the bioinformatics studies (that is,
n=0-4), to assess whether they retained the phase behavior
characteristic of the canonical Val-Pro-Gly-X-Gly (SEQ ID NO: 3)
motif found in tropoelastin. FIG. 21 confirms that all these new
arrangements of Pro and Gly residues are conducive to "smart"
biopolymers with stimuli-responsive, phase transition behavior
analogous to that of tropoelastin. These protein-polymers appear to
have similar secondary structure propensities as elastin-like
polypeptides and display an ensemble of highly dynamic conformers
characteristic of other IDPs (FIG. 21C). This exciting finding
significantly relaxes the sequence constraints on these polymers,
as it allows for the incorporation of a number of short motifs
within the PX.sub.nG unit or in the surrounding residues--10
residues between PX.sub.nG units are permissible. For instance, a
number of neuroactive proteins present their bioactive sequences as
tandem repeats embedded within PX.sub.4G units. Pro-rich proteins
use tandem repeats of PX.sub.4G motifs for the presentation of
bioactive peptides. The two neuroactive proteins FARXamide-related
neuropeptides and PRQFVamide display tandem repeats of the
bioactive peptides PFLRF (SEQ ID NO: 108) and PRQFV (SEQ ID NO:
109) embedded within PX.sub.4G motifs. A similar localized region
of PX.sub.4G units was observed with highly conserved X.sub.4
residues in a transcription factor from yeast (SPTS).
[0106] It is also possible to identify "smart" biopolymers with
three types of phase behavior marked by three degrees of hysteresis
in the reversibility of their thermally-triggered phase transition:
i) zero, ii) finite, and iii) heat-sensitive infinite hysteresis
(FIG. 21D). The "heat-sensitivity" of the latter category refers to
the finding that these protein-polymers display zero hysteresis
below a critical threshold temperature (typically around 40.degree.
C. for the polymers herein synthesized). Six different
environmentally responsive polypeptides (VGAPVG).sub.35,
(VGPVG).sub.30, (VGPAVG).sub.20, (VPGAVG).sub.30, (VTPAVG).sub.25,
and (TPVAVG).sub.30, (repeats of SEQ ID NOs: 24, 98, 17, 38, 18 and
31 respectively) were found to show irreversible phase separation
when heated to 75.degree. C., whereas they displayed reversible
phase transition behavior if heated below a given threshold
temperature. The (TPVAVG).sub.30 (SEQ ID NO: 31) polypeptide was
purified by exploiting the reversibility of its phase behavior in
response to changes in ionic strength. Environmentally responsive
polypeptides with the repeat unit (VTPAVG) (SEQ ID NO: 18)
exhibited a very complex phase transition behavior as they
displayed both heat-sensitive infinite hysteresis and finite
hysteresis below the threshold temperature. The phase behavior was
characterized in PBS at a polypeptide concentration of 50
.mu.M.
[0107] The heat-sensitive, infinite hysteresis of the
environmentally responsive polypeptides was found to arise from the
emergence of ordered secondary structures that stabilized the
insoluble phase. Whereas an environmentally responsive polypeptide
that displayed finite hysteresis rapidly recovered its
conformational disorder on lowering the temperature below the phase
transition temperature adjusted by its degree of hysteresis,
environmentally responsive polypeptides with heat-sensitive
infinite hysteresis underwent conformational changes that persisted
on cooling. An environmentally responsive polypeptide that
displayed such a large hysteresis did not exhibit observable
reversibility below any given temperature threshold, and displayed
ordering on coacervation. Turbidity and CD data were acquired in
water at a polypeptide concentration of 5 .mu.M.
[0108] The emergence of more ordered secondary structures on phase
transition was identified as a primary factor responsible of the
increasing degree of hysteresis. Despite the respective degree of
hysteresis, which provides a tool for the biomedical and
biotechnological exploitation of these materials, the reversible
phase transition behavior of all protein-polymers herein described
enabled their purification by recursive rounds of phase
separation.
[0109] Having relaxed the constraints on the distribution of Pro
and Gly residues in these protein-polymers, sequence diversity was
maximized. The bioinformatics studies underscored the role of
overall hydropathicity over local biases in hydrophobicity.
Protein-polymers were designed with a target, average hydropathy,
but incorporating amino acids with a wide distribution of
hydropathies. FIG. 22A shows the generation of protein-polymers
composed of hexapeptide motifs wherein only one Pro and one Gly
residue are fixed and all residues are otherwise randomized. A
target hydropathy of 37.degree. C. was selected to generate a
protein-sized biopolymer (240 resides in length) with a widely
diverse amino acid composition spanning the entire range of
hydropathies (FIG. 22B).
[0110] The primary structure of the randomized environmentally
responsive polypeptide reported in FIGS. 22A-22K was of the form
(Z-Z-P-X-G-Z).sub.40 (SEQ ID NO: 11).sub.40 and had the following
sequence: [0111]
SKGPGVPAGHRYPIGGGQPHGKGCPDGVFRPVGLGAPYGHGAPNGMHRPLGIGKPRGH
MYPKGQGQPMGHLVPDGVGFPRGRKKPVGVGKPIGNGHPIGARTPLGYGMPDGVGMP
MGLFLPNGHGAPHGQGYPAGKLIPKGKGHPFGKGRPLGAGRPTGFKMPKGLGKPMGVG
QPQGHFVPFGLGQPTGQGAPRGGSQPAGLGHPLGAGAPAGRCHPYGMGVPRGLAMPRG
HGQPRGVGYPKGHGWP) (SEQ ID NO: 105). The amino acid sequence
included the N terminal peptide SKGP (SEQ ID NO: 106) and the
C-terminal tripeptide GWP. The Z and X residues were randomized
using a normal distribution with a mean hydropathy of 37.degree. C.
and a standard deviation of 50.degree. C.--in order to ensure large
sequence diversity.
[0112] This protein-polymer behaved as an IDP (FIG. 22C) and
displayed phase transition behavior (FIG. 22D). Surprisingly, the
target hydropathy was a good predictor of the temperature at which
the biopolymer underwent phase separation (.about.40.degree. C.).
This protein-polymer lacked any repeating motif (unlike
tropoelastin or resilin), and Pro-X-Gly units were quite rare in
all proteins that we analyzed. A "random" biopolymer was designed
the size of a short protein-domain (30 amino acids in length).
Polymers of this domain displayed phase transition behavior (FIG.
24). Although Gly enrichment observed in the surroundings of the
Pro-X.sub.n-Gly units for most non-fibrillar Pro and Gly-rich
proteins was incorporated, motifs were identified that demonstrate
that such Gly enrichment is not necessary for the design of "smart"
biopolymers with fully reversible phase transition behavior (FIG.
25).
[0113] Gly has a role in modulating the assembly behavior of IDPs.
The phase behavior of two protein-polymers were studied wherein Gly
was placed preceding a Pro-Gly and a Pro-X-X-Gly unit, and their
behavior compared with those of identical motifs where this Gly
residue was mutated. Positioning of a Gly residue N-terminal to a
Pro-X.sub.n-Gly motif enhances the propensity for coacervation--by
decreasing the transition temperature (FIG. 22E-F)--, promotes the
formation of irreversible aggregates (FIG. 22E) and leads to
changes in assembly behavior (inset of FIG. 22F). This Gly-induced
instability was also evidenced on the insoluble expression of these
protein-polymers (data not shown), despite their high solubility in
PBS once purified. These results underline the role of Gly as a
potent modulator of the assembly of IDPs such that this modulatory
role may be exploited for the synthesis of "smart" biopolymers that
reproduce the assembly behavior and/or mechanical properties of
collagen and silks.
[0114] The complex phase behaviors indicate a relationship between
the syntax of the protein-polymer, its secondary structure and its
phase behavior. These structure-function relationships are
characteristic of folded proteins. IDPs exhibit highly flexible
backbone conformations, but are unlikely to be true random coils.
To demonstrate that the polypeptide conformation--as opposed to
composition--exerts a potent modulatory role that cannot be
explained by a simple random coil model of the disordered state of
these polymers, the effect of backbone reversal on phase behavior
was studied. Backbone reversal produces a biopolymer that has an
identical sequence as the parent biopolymer if read from the C- to
the N-terminus, thus having identical hydrophobicity and identical
distribution of amino acids (FIG. 22G). However, whereas folded
proteins often lose their structure and function on backbone
reversal, the effect of backbone reversal on the function (that is,
phase behavior) of Pro- and Gly-rich polymers that are
intrinsically disordered was unexpectedly found to lead to changes
in function (FIG. 21H-J and FIG. 26A), which result from changes in
the ensemble of conformations that describe the dynamic backbone of
these polymers (FIG. 21K and FIG. 26B-D). This finding suggests
that these proteins are unlike synthetic polymers that exist as
random coils, and are protein-polymers that are intrinsically
disordered. The recurrent link between intrinsic disorder and
"smart" behavior suggests that a polypeptide with intrinsic
disorder displays "smart" behavior.
[0115] Environmentally responsive polypeptides with truly
protein-like complexity may exert a biological function encoded in
their sequence. Their syntax may also be compatible with sequences
that have defined secondary structure propensities as these abound
in most proteins. Environmentally responsive polypeptides were made
with a repeating unit based on the matrikine motif Gly-X-X-Pro-Gly
(SEQ ID NO: 21) (FIG. 4A)--encoding bioactive motifs released on
cleavage of various extracellular matrix proteins--, unlike
environmentally responsive polypeptides with a disrupted motif but
identical composition and phase behavior (FIG. 4B), are capable of
preventing tumor growth in a mouse model (FIG. 4C). An
environmentally responsive polypeptide inspired in the matrikine
motif GXXPG (SEQ ID NO: 21), SM1-24 (250 .mu.M in PBS), was found
to prevent the grafting of 1.times.10.sup.6HT1080 tumor cells
inoculated into the back of nude mice (FIG. 27). A control
polypeptide, SM2-24 (250 .mu.M in PBS), with a disrupted motif but
identical phase transition behavior (FIG. 23) had no effect on
tumor growth. Tumor volumes were measured 19 days after
inoculation.
[0116] An environmentally responsive polypeptide was synthesized
based on the bioactive site of murine Endostatin, and was found to
displays an inverse phase transition temperature reminiscent of
other environmentally responsive polypeptides with simpler syntax
(FIG. 38). The phase transition of a 5 .mu.M solution of mEndo1-6
in PBS (pH 6.4) (A) was accompanied by a decrease in the disorder
of the polypeptide conformation (B), as measured by circular
dichroism under identical conditions as in (A).
[0117] The diversity of the ensemble of structures observed in our
environmentally responsive polypeptides are compatible with protein
domains that have very defined local secondary structure
propensities. Environmentally responsive polypeptides were
synthesized that were composed of the bioactive domains of
endostatin from humans and mice, which are well folded protein
domains (25-27 amino acids in length) that retain the potent
anti-angiogenic activity of endostatin as isolated peptides (FIG.
23D). These environmentally responsive polypeptides that behave as
IDPs (FIG. 23E) and display "smart" behavior (FIG. 23F). This is
surprising given the partially folded nature of these domains in
the native protein (FIG. 23D), the existence of significant regions
with high propensities to fold into .alpha.-helices and
.beta.-sheets in the polymerized polypeptides (FIG. 23G), and the
relatively low Pro and Gly content (10% Pro and 14-17% Gly,
compared with 20% Pro and 40% Gly in elastin-like polypeptides).
Peptide hormones, which often have similar, local secondary
structure propensities (FIG. 4G) may be designed and formed from
environmentally responsive polypeptides described herein.
[0118] Environmentally responsive polypeptides were also made that
showed UCST behaviour (FIG. 28). Such polypeptides were found to
display reversible UCST behavior in PBS which behavior may be tuned
by polypeptide concentration and the number of repeating units.
(FIG. 29).
[0119] Environmentally responsive polypeptides incorporating the
peptide drug GRGDSP were found to be bioactive and their
bioactivity could be switched on or off by their phase transition
behavior. (FIG. 30; Left panel) Whereas increasing concentrations
of (GRGDSPYG)-12 (SEQ ID NO: 44).sub.12, which has an UCST below
37.degree. C. and is thus soluble, prevented cell adhesion of
PC3-luc-C6 cells after a 3 h treatment, (GRGDSPYG)-20 (SEQ ID NO:
44).sub.20, which displayed an UCST above 37.degree. C., had almost
no effect on cell adhesion as its concentration increases. (FIG.
30; Right panel) These bioactive environmentally responsive
polypeptides were not toxic to the cells as they remained viable
when given sufficient time to adhere--perhaps through mechanisms
that are not dependent on the integrins targeted by GRGDSP. Control
cultures that matched the experimental treatment with the maximum
concentration of residual Urea ((GRGDSPYG)-12 (SEQ ID NO:
44).sub.12) demonstrated that residual Urea (up to 0.1 M for the 40
.mu.M samples) did not affect cell adhesion or viability.
[0120] The UCST behavior of environmentally responsive polypeptides
containing RGD tripeptides exhibited a complex response to buffer
ionic strength, wherein small concentrations of salt decreased the
UCST (due to charge screening) and high concentrations had the
opposite effect as they favored hydrophobic interactions (FIG.
31).
[0121] The UCST behavior of environmentally responsive polypeptides
was modulated by electrostatic interactions between positively and
negatively charged amino acids within the sequence (FIG. 32). At pH
2.0, aspartic acid was fully protonated, which largely increased
the hydrophobicity of (GRGDSPYG)-20 ((SEQ ID NO: 44).sub.20), and
yet, instead of observing an increase in its UCST (as it would be
expected), a drastic reduction in the UCST was observed that
demonstrates the role of electrostatic interactions--here between
Arg and Asp residues--in increasing the UCST of these
biopolymers.
[0122] The UCST behavior of environmentally responsive polypeptides
did not require electrostatic interactions (FIG. 33). The aspartic
acid in RGD-containing polypeptides was substituted with asparagine
and did not eliminate the UCST behavior displayed by these
biopolymers. These biopolymers when tested may also have displayed
LCST behavior.
[0123] The design of RGD-containing environmentally responsive
polypeptides that display UCST behavior was found to be compatible
with multiple arrangements of Pro and Gly residues. A PG dipeptide,
instead of a P-Y-G tripeptide, did not perturb the UCST behavior
(FIG. 34). The UCST behavior of the polypeptides was tuned by
adjusting the hydrophobicity of the residues comprising the
repeating unit. Substituting a Gly residue by a more hydrophilic
residue, glutamine, significantly reduced the UCST of the
biopolymer (FIG. 35).
[0124] Environmentally responsive polypeptides that contain the
peptide drug PHSRN (SEQ ID NO: 107) were also found to display UCST
behavior (FIG. 36). These biopolymers exhibited reversible phase
behavior (left) and were intrinsically disordered (right). The UCST
behavior of the polypeptides thus arose from residues capable of
intermolecular hydrogen bonding (that is, arginine and serine).
[0125] Environmentally responsive polypeptides were designed to
display complex phase behaviors wherein the biopolymers display
both UCST and LCST, and the LCST is lower than the UCST. (FIG. 37).
Environmentally responsive polypeptides with composite motifs
composed of one UCST motif and one LCST motif would enable the
design of biopolymers that display both UCST and LCST if the LCST
is greater than the UCST. Tuning this band-pass behavior will
enable the design of environmentally responsive polypeptides that
display phase separation only in a very narrow temperature window,
which would facilitate the manipulation of complex mixtures of
environmentally responsive polypeptides, such as in applications
involving multiplexing.
[0126] Any patents or publications mentioned in this specification
are herein incorporated by reference in their entirety to the same
extent as if each individual publication was specifically and
individually indicated to be incorporated by reference.
[0127] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods, procedures,
treatments, molecules, and specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the claims.
Sequence CWU 1
1
13715PRTArtificial SequenceSynthetic Peptidemisc_feature(5)..(5)Xaa
can be any naturally occurring amino acid 1Gly Pro Gly Gly Xaa 1 5
25PRTArtificial SequenceSynthetic Peptide 2Gly Pro Gly Gln Gln 1 5
35PRTArtificial SequenceSynthetic Peptidemisc_feature(4)..(4)Xaa
can be any naturally occurring amino acid 3Val Pro Gly Xaa Gly 1 5
45PRTArtificial SequenceSynthetic Peptidemisc_feature(4)..(4)Xaa
can be any naturally occurring amino acid 4Leu Pro Gly Xaa Gly 1 5
55PRTArtificial SequenceSynthetic Peptidemisc_feature(4)..(4)Xaa
can be any naturally occurring amino acid 5Ile Pro Gly Xaa Gly 1 5
65PRTArtificial SequenceSynthetic Peptide 6Val Pro Ala Val Gly 1 5
75PRTArtificial SequenceSynthetic Peptidemisc_feature(4)..(5)Xaa
can be any naturally occurring amino acid 7Gly Pro Gly Xaa Xaa 1 5
86PRTArtificial SequenceSynthetic Peptide 8Ala Val Pro Gly Val Gly
1 5 96PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(6)Xaa can be any naturally occurring amino
acid 9Xaa Xaa Pro Gly Xaa Xaa 1 5 106PRTArtificial
SequenceSynthetic Peptide 10Thr Val Pro Gly Ala Gly 1 5
116PRTArtificial SequenceSynthetic Peptidemisc_feature(1)..(2)Xaa
can be any naturally occurring amino acidmisc_feature(4)..(4)Xaa
can be any naturally occurring amino acidmisc_feature(6)..(6)Xaa
can be any naturally occurring amino acid 11Xaa Xaa Pro Xaa Gly Xaa
1 5 1230PRTArtificial SequenceSynthetic Peptide 12Gly Ala Pro Phe
Gly Phe Ala Ile Pro Met Gly Ala Gly Phe Pro Thr 1 5 10 15 Gly Gly
Leu Ala Pro Phe Gly Met Gly Leu Pro Ala Gly Met 20 25 30
137PRTArtificial SequenceSynthetic Peptidemisc_feature(1)..(2)Xaa
can be any naturally occurring amino acidmisc_feature(4)..(4)Xaa
can be any naturally occurring amino acidmisc_feature(6)..(7)Xaa
can be any naturally occurring amino acid 13Xaa Xaa Pro Xaa Gly Xaa
Xaa 1 5 148PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(8)Xaa can be any naturally occurring amino
acid 14Xaa Xaa Pro Xaa Xaa Gly Xaa Xaa 1 5 1510PRTArtificial
SequenceSynthetic Peptidemisc_feature(1)..(2)Xaa can be any
naturally occurring amino acidmisc_feature(4)..(7)Xaa can be any
naturally occurring amino acidmisc_feature(9)..(10)Xaa can be any
naturally occurring amino acid 15Xaa Xaa Pro Xaa Xaa Xaa Xaa Gly
Xaa Xaa 1 5 10 164PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(3)Xaa can be any naturally occurring amino
acid 16Pro Xaa Xaa Gly 1 176PRTArtificial SequenceSynthetic Peptide
17Val Gly Pro Ala Val Gly 1 5 186PRTArtificial SequenceSynthetic
Peptide 18Val Thr Pro Ala Val Gly 1 5 196PRTArtificial
SequenceSynthetic Peptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 19Val Pro Xaa Gly Xaa Gly 1 5
206PRTArtificial SequenceSynthetic Peptidemisc_feature(1)..(1)Xaa
can be any naturally occurring amino acidmisc_feature(5)..(5)Xaa
can be any naturally occurring amino acid 20Xaa Val Pro Gly Xaa Gly
1 5 215PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(3)Xaa can be any naturally occurring amino
acid 21Gly Xaa Xaa Pro Gly 1 5 226PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(2)Xaa can be any naturally occurring amino
acidMISC_FEATURE(4)..(4)Xaa is from 1 to 4 amino acids that are not
P or Gmisc_feature(6)..(6)Xaa can be any naturally occurring amino
acid 22Xaa Xaa Pro Xaa Gly Xaa 1 5 237PRTArtificial
SequenceSynthetic Peptidemisc_feature(1)..(2)Xaa can be any
naturally occurring amino acidMISC_FEATURE(4)..(4)Xaa is from 1 to
4 amino acids that are not P or Gmisc_feature(6)..(7)Xaa can be any
naturally occurring amino acid 23Xaa Xaa Pro Xaa Gly Xaa Xaa 1 5
246PRTArtificial SequenceSynthetic Peptide 24Val Gly Ala Pro Val
Gly 1 5 256PRTArtificial SequenceSynthetic Peptide 25Leu Gly Ala
Pro Val Gly 1 5 269PRTArtificial SequenceSynthetic Peptide 26Val
Pro Ser Ala Leu Tyr Gly Val Gly 1 5 275PRTArtificial
SequenceSynthetic Peptide 27Val Ala Pro Val Gly 1 5
286PRTArtificial SequenceSynthetic Peptide 28Val Thr Pro Ala Val
Gly 1 5 299PRTArtificial SequenceSynthetic Peptide 29Val Pro Ser
Asp Asp Tyr Gly Gln Gly 1 5 309PRTArtificial SequenceSynthetic
Peptide 30Val Pro Ser Asp Asp Tyr Gly Val Gly 1 5 316PRTArtificial
SequenceSynthetic Peptide 31Thr Pro Val Ala Val Gly 1 5
329PRTArtificial SequenceSynthetic Peptide 32Val Pro Ser Thr Asp
Tyr Gly Val Gly 1 5 336PRTArtificial SequenceSynthetic Peptide
33Val Pro Ala Gly Val Gly 1 5 346PRTArtificial SequenceSynthetic
Peptide 34Val Pro Thr Gly Val Gly 1 5 356PRTArtificial
SequenceSynthetic Peptide 35Val Pro Ala Gly Leu Gly 1 5
365PRTArtificial SequenceSynthetic Peptide 36Val Pro His Val Gly 1
5 376PRTArtificial SequenceSynthetic Peptide 37Val His Pro Gly Val
Gly 1 5 386PRTArtificial SequenceSynthetic Peptide 38Val Pro Gly
Ala Val Gly 1 5 396PRTArtificial SequenceSynthetic Peptide 39Val
Pro Gly Val Ala Gly 1 5 405PRTArtificial SequenceSynthetic Peptide
40Val Arg Pro Val Gly 1 5 417PRTArtificial SequenceSynthetic
Peptide 41Gly Arg Gly Asp Ser Pro Tyr 1 5 427PRTArtificial
SequenceSynthetic Peptide 42Gly Arg Gly Asp Ser Pro His 1 5
437PRTArtificial SequenceSynthetic Peptide 43Gly Arg Gly Asp Ser
Pro Val 1 5 448PRTArtificial SequenceSynthetic Peptide 44Gly Arg
Gly Asp Ser Pro Tyr Gly 1 5 457PRTArtificial SequenceSynthetic
Peptide 45Arg Pro Leu Gly Tyr Asp Ser 1 5 467PRTArtificial
SequenceSynthetic Peptide 46Arg Pro Ala Gly Tyr Asp Ser 1 5
477PRTArtificial SequenceSynthetic Peptide 47Gly Arg Gly Asp Ser
Tyr Pro 1 5 488PRTArtificial SequenceSynthetic Peptide 48Gly Arg
Gly Asp Ser Pro Tyr Gln 1 5 498PRTArtificial SequenceSynthetic
Peptide 49Gly Arg Gly Asn Ser Pro Tyr Gly 1 5 508PRTArtificial
SequenceSynthetic Peptide 50Gly Arg Gly Asp Ala Pro Tyr Gln 1 5
518PRTArtificial SequenceSynthetic Peptide 51Val Pro His Ser Arg
Asn Gly Gly 1 5 528PRTArtificial SequenceSynthetic Peptide 52Val
Pro His Ser Arg Asn Gly Leu 1 5 5328PRTArtificial SequenceSynthetic
Peptide 53Val Pro Gly His Ser His Arg Asp Phe Gln Pro Val Leu His
Leu Val 1 5 10 15 Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly
20 25 5430PRTArtificial SequenceSynthetic Peptide 54His Thr His Gln
Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Thr Pro
Leu Ser Gly Gly Met Arg Gly Ile Arg Pro Gly Gly 20 25 30
5512PRTArtificial SequenceSynthetic Peptide 55Phe Glu Trp Thr Pro
Gly Trp Tyr Gln Pro Tyr Gly 1 5 10 566PRTArtificial
SequenceSynthetic Peptidemisc_feature(1)..(2)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 56Xaa Xaa Pro Gly Xaa Gly 1 5
578PRTArtificial SequenceSynthetic Peptidemisc_feature(7)..(7)Xaa
can be any naturally occurring amino acid 57Gly Arg Gly Asp Ser Pro
Xaa Gly 1 5 588PRTArtificial SequenceSynthetic
Peptidemisc_feature(8)..(8)Xaa can be any naturally occurring amino
acid 58Gly Arg Gly Asp Ser Pro Gly Xaa 1 5 5911PRTArtificial
SequenceSynthetic Peptide 59Phe Glu Trp Thr Pro Gly Trp Tyr Gln Pro
Tyr 1 5 10 605PRTArtificial SequenceSynthetic Peptide 60Glu Ile Leu
Asp Val 1 5 614PRTArtificial SequenceSynthetic Peptide 61Arg Glu
Asp Val 1 625PRTArtificial SequenceSynthetic Peptide 62Met Ser Lys
Gly Pro 1 5 637PRTArtificial SequenceSynthetic Peptide 63His His
His His His His Tyr 1 5 6436PRTArtificial SequenceSynthetic Peptide
64Ala Val Pro Gly Val Gly Ala Val Pro Gly Val Gly Ala Val Pro Gly 1
5 10 15 Val Gly Ala Val Pro Gly Val Gly Ala Val Pro Gly Val Gly Cys
Val 20 25 30 Pro Gly Val Gly 35 6536PRTArtificial SequenceSynthetic
Peptide 65Val Pro Ala Gly Val Gly Val Pro Ala Gly Val Gly Val Pro
Ala Gly 1 5 10 15 Val Gly Val Pro Ala Gly Val Gly Val Pro Ala Gly
Val Gly Val Pro 20 25 30 Cys Gly Val Gly 35 666PRTArtificial
SequenceSynthetic Peptidemisc_feature(2)..(5)Xaa can be any
naturally occurring amino acid 66Pro Xaa Xaa Xaa Xaa Gly 1 5
676PRTArtificial SequenceSynthetic Peptide 67Val Ala Pro Gly Val
Gly 1 5 686PRTArtificial SequenceSynthetic Peptide 68Gly Val Pro
Gly Ala Val 1 5 696PRTArtificial SequenceSynthetic Peptide 69Gly
Val Pro Gly Val Ala 1 5 706PRTArtificial SequenceSynthetic Peptide
70Thr Val Pro Gly Val Gly 1 5 716PRTArtificial SequenceSynthetic
Peptide 71Thr Val Pro Gly Ala Gly 1 5 726PRTArtificial
SequenceSynthetic Peptide 72Gly Ala Pro Gly Val Gly 1 5
736PRTArtificial SequenceSynthetic Peptide 73Ala Val Pro Gly Val
Ala 1 5 746PRTArtificial SequenceSynthetic Peptide 74Gly Ala Pro
Gly Gly Gly 1 5 756PRTArtificial SequenceSynthetic Peptide 75Gly
Ala Pro Gly Ala Gly 1 5 766PRTArtificial SequenceSynthetic Peptide
76Gly Gly Pro Gly Ala Gly 1 5 777PRTArtificial SequenceSynthetic
Peptide 77Gly Val Pro Ala Gly Val Gly 1 5 787PRTArtificial
SequenceSynthetic Peptide 78Val Gly Pro Val Gly Val Gly 1 5
797PRTArtificial SequenceSynthetic Peptide 79Gly Val Pro Thr Gly
Val Gly 1 5 807PRTArtificial SequenceSynthetic Peptide 80Gly Ala
Pro Val Gly Val Gly 1 5 817PRTArtificial SequenceSynthetic Peptide
81Val Ala Pro Val Gly Val Ala 1 5 827PRTArtificial
SequenceSynthetic Peptide 82Gly Ala Pro Phe Gly Phe Ala 1 5
837PRTArtificial SequenceSynthetic Peptide 83Ala Ile Pro Met Gly
Ala Gly 1 5 847PRTArtificial SequenceSynthetic Peptide 84Gly Phe
Pro Thr Gly Gly Leu 1 5 857PRTArtificial SequenceSynthetic Peptide
85Leu Ala Pro Phe Gly Met Gly 1 5 867PRTArtificial
SequenceSynthetic Peptide 86Gly Leu Pro Ala Gly Met Gly 1 5
877PRTArtificial SequenceSynthetic Peptide 87Gly Val Pro Ala Val
Gly Val 1 5 887PRTArtificial SequenceSynthetic Peptide 88Gly Val
Pro His Val Gly Val 1 5 897PRTArtificial SequenceSynthetic Peptide
89Val Gly Pro Ala Val Gly Val 1 5 907PRTArtificial
SequenceSynthetic Peptide 90Val Thr Pro Ala Val Gly Val 1 5
9110PRTArtificial SequenceSynthetic Peptide 91Gly Val Pro Ser Ala
Leu Tyr Gly Val Gly 1 5 10 9210PRTArtificial SequenceSynthetic
Peptide 92Gly Val Pro Ser Asp Asp Tyr Gly Gln Gly 1 5 10
9310PRTArtificial SequenceSynthetic Peptide 93Gly Val Pro Ser Asp
Asp Tyr Gly Val Gly 1 5 10 946PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(5)Xaa can be any naturally occurring amino
acid 94Pro Xaa Xaa Xaa Xaa Gly 1 5 955PRTArtificial
SequenceSynthetic PeptideMISC_FEATURE(1)..(1)Xaa is 4 or less amino
acidsMISC_FEATURE(3)..(3)Xaa is 4 or less amino
acidsMISC_FEATURE(5)..(5)Xaa is 4 or less amino acids 95Xaa Pro Xaa
Gly Xaa 1 5 965PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(4)Xaa can be any naturally occurring amino
acid 96Pro Xaa Xaa Xaa Gly 1 5 975PRTArtificial SequenceSynthetic
Peptide 97Val Pro Gly Val Gly 1 5 985PRTArtificial
SequenceSynthetic Peptide 98Val Gly Pro Val Gly 1 5
995PRTArtificial SequenceSynthetic Peptide 99Ala Pro Gly Val Gly 1
5 1004PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(1)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(4)Xaa can be any naturally occurring amino
acid 100Xaa Pro Gly Xaa 1 10130PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(4)Xaa can be any naturally occurring amino
acidmisc_feature(6)..(8)Xaa can be any naturally occurring amino
acidmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidmisc_feature(12)..(14)Xaa can be any naturally occurring amino
acidmisc_feature(16)..(16)Xaa can be any naturally occurring amino
acidmisc_feature(18)..(20)Xaa can be any naturally occurring amino
acidmisc_feature(22)..(22)Xaa can be any naturally occurring amino
acidmisc_feature(24)..(26)Xaa can be any naturally occurring amino
acidmisc_feature(28)..(28)Xaa can be any naturally occurring amino
acidmisc_feature(30)..(30)Xaa can be any naturally occurring amino
acid 101Xaa Xaa Pro Xaa Gly Xaa Xaa Xaa Pro Xaa Gly Xaa Xaa Xaa Pro
Xaa 1 5 10 15 Gly Xaa Xaa Xaa Pro Xaa Gly Xaa Xaa Xaa Pro Xaa Gly
Xaa 20 25 30 1024PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(3)Xaa can be any naturally occurring amino
acid 102Pro Xaa Xaa Gly 1 10311PRTArtificial SequenceSynthetic
Peptide 103Ala Gln Thr Pro Ser Ser Gln Tyr Gly Ala Pro 1 5 10
1044PRTArtificial SequenceSynthetic Peptide 104Tyr Gly Ala Pro 1
105247PRTArtificial SequenceSynthetic Peptide 105Ser Lys Gly Pro
Gly Val Pro Ala Gly His Arg Tyr Pro Ile Gly Gly 1 5 10 15 Gly Gln
Pro His Gly Lys Gly Cys Pro Asp Gly Val Phe Arg Pro Val 20 25 30
Gly Leu Gly Ala Pro Tyr Gly His Gly Ala Pro Asn Gly Met His Arg 35
40 45 Pro Leu Gly Ile Gly Lys Pro Arg Gly His Met Tyr Pro Lys Gly
Gln 50 55 60 Gly Gln Pro Met Gly His Leu Val Pro Asp Gly Val Gly
Phe Pro Arg 65 70 75 80 Gly Arg Lys Lys Pro Val Gly Val Gly Lys Pro
Ile Gly Asn Gly His 85 90 95 Pro Ile Gly Ala Arg Thr Pro Leu Gly
Tyr Gly Met Pro Asp Gly Val 100 105 110 Gly Met Pro Met Gly Leu Phe
Leu Pro Asn Gly His Gly Ala Pro His 115 120 125 Gly Gln Gly Tyr Pro
Ala Gly Lys Leu Ile Pro Lys Gly Lys Gly His 130 135 140 Pro Phe Gly
Lys Gly Arg Pro Leu Gly Ala Gly Arg Pro Thr Gly Phe 145 150 155 160
Lys Met Pro Lys Gly Leu Gly Lys Pro Met Gly Val Gly Gln Pro Gln 165
170 175 Gly His Phe Val Pro Phe Gly Leu Gly Gln Pro Thr Gly Gln Gly
Ala 180 185 190 Pro Arg Gly Gly Ser Gln Pro Ala Gly Leu Gly His Pro
Leu Gly Ala 195 200 205 Gly Ala Pro Ala Gly Arg Cys His Pro Tyr Gly
Met Gly Val Pro Arg 210 215 220 Gly Leu Ala Met Pro Arg Gly His Gly
Gln Pro Arg Gly Val Gly Tyr 225 230 235 240 Pro Lys Gly His Gly Trp
Pro 245 1064PRTArtificial SequenceSynthetic Peptide 106Ser Lys Gly
Pro 1 1075PRTArtificial SequenceSynthetic Peptide 107Pro His Ser
Arg Asn 1 5 1085PRTArtificial SequenceSynthetic Peptide 108Pro Phe
Leu Arg Phe 1 5 1095PRTArtificial SequenceSynthetic Peptide 109Pro
Arg Gln Phe Val 1 5 110241PRTArtificial SequenceSynthetic Peptide
110Gly Val Gly Pro Ala Gly His Arg Tyr Pro Ile Gly Gly Gln Gly Pro
1 5
10 15 His Gly Lys Cys Gly Pro Asp Gly Val Phe Arg Pro Val Gly Leu
Ala 20 25 30 Gly Pro Tyr Gly His Ala Gly Pro Asn Gly Met His Arg
Pro Leu Gly 35 40 45 Ile Lys Gly Pro Arg Gly His Met Tyr Pro Lys
Gly Gln Gln Gly Pro 50 55 60 Met Gly His Leu Val Pro Asp Gly Val
Phe Gly Pro Arg Gly Arg Lys 65 70 75 80 Lys Pro Val Gly Val Lys Gly
Pro Ile Gly Asn His Gly Pro Ile Gly 85 90 95 Ala Arg Thr Pro Leu
Gly Tyr Met Gly Pro Asp Gly Val Met Gly Pro 100 105 110 Met Gly Leu
Phe Leu Pro Asn Gly His Ala Gly Pro His Gly Gln Tyr 115 120 125 Gly
Pro Ala Gly Lys Leu Ile Pro Lys Gly Lys His Gly Pro Phe Gly 130 135
140 Lys Arg Gly Pro Leu Gly Ala Arg Gly Pro Thr Gly Phe Lys Met Pro
145 150 155 160 Lys Gly Leu Lys Gly Pro Met Gly Val Gln Gly Pro Gln
Gly His Phe 165 170 175 Val Pro Phe Gly Leu Gln Gly Pro Thr Gly Gln
Ala Gly Pro Arg Gly 180 185 190 Gly Ser Gln Pro Ala Gly Leu His Gly
Pro Leu Gly Ala Ala Gly Pro 195 200 205 Ala Gly Arg Cys His Pro Tyr
Gly Met Val Gly Pro Arg Gly Leu Ala 210 215 220 Met Pro Arg Gly His
Gln Gly Pro Arg Gly Val Tyr Gly Pro Lys Gly 225 230 235 240 His
1115PRTArtificial SequenceSynthetic Peptide 111Val Pro Gly Val Ala
1 5 1126PRTArtificial SequenceSynthetic Peptide 112Val Ala Pro Gly
Ala Gly 1 5 1138PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(1)Xaa can be any naturally occurring amino
acidMISC_FEATURE(3)..(3)Xaa is 0 to 4 amino acids that are not P or
Gmisc_feature(5)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(8)..(8)Xaa can be any naturally occurring amino
acid 113Xaa Pro Xaa Gly Xaa Arg Gly Xaa 1 5 1146PRTArtificial
SequenceSynthetic Peptide 114Ala Pro Val Gly Val Gly 1 5
1156PRTArtificial SequenceSynthetic Peptide 115Ala Pro Val Gly Leu
Gly 1 5 1165PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 116Val Xaa Pro Val Gly 1 5 1176PRTArtificial SequenceSynthetic
Peptidemisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 117Val Xaa Pro Ala Val Gly 1 5 1185PRTArtificial
SequenceSynthetic Peptide 118Gly Val Ala Pro Val 1 5
1195PRTArtificial SequenceSynthetic Peptide 119Gly Val Gly Pro Val
1 5 1206PRTArtificial SequenceSynthetic Peptide 120Gly Val Gly Ala
Pro Val 1 5 12130PRTArtificial SequenceSynthetic Peptide 121Gly Ala
Val Pro Gly Val Gly Ala Val Pro Gly Val Gly Ala Val Pro 1 5 10 15
Gly Val Gly Ala Val Pro Gly Val Gly Cys Val Pro Gly Val 20 25 30
12230PRTArtificial SequenceSynthetic Peptide 122Gly Val Pro Ala Gly
Val Gly Val Pro Ala Gly Val Gly Val Pro Ala 1 5 10 15 Gly Val Gly
Val Pro Ala Gly Val Gly Val Pro Cys Gly Val 20 25 30
12356PRTArtificial SequenceSynthetic Peptide 123Val Pro Gly His Ser
His Arg Asp Phe Gln Pro Val Leu His Leu Val 1 5 10 15 Ala Leu Asn
Ser Pro Leu Ser Gly Gly Met Arg Gly Val Pro Gly His 20 25 30 Ser
His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn Ser 35 40
45 Pro Leu Ser Gly Gly Met Arg Gly 50 55 12460PRTArtificial
SequenceSynthetic Peptide 124His Thr His Gln Asp Phe Gln Pro Val
Leu His Leu Val Ala Leu Asn 1 5 10 15 Thr Pro Leu Ser Gly Gly Met
Arg Gly Ile Arg Pro Gly Gly His Thr 20 25 30 His Gln Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn Thr Pro 35 40 45 Leu Ser Gly
Gly Met Arg Gly Ile Arg Pro Gly Gly 50 55 60 12574PRTArtificial
SequenceSynthetic Peptide 125Asp Val Ser Thr Pro Pro Thr Val Leu
Pro Asp Asn Phe Pro Arg Tyr 1 5 10 15 Pro Val Gly Lys Phe Phe Gln
Tyr Asp Thr Trp Lys Gln Ser Thr Gln 20 25 30 Arg Leu Pro Gly Gly
Asp Val Ser Thr Pro Pro Thr Val Leu Pro Asp 35 40 45 Asn Phe Pro
Arg Tyr Pro Val Gly Lys Phe Phe Gln Tyr Asp Thr Trp 50 55 60 Lys
Gln Ser Thr Gln Arg Leu Pro Gly Gly 65 70 12652PRTArtificial
SequenceSynthetic Peptide 126Phe Asn Ala Pro Phe Asp Val Gly Ile
Lys Leu Ser Gly Val Gln Tyr 1 5 10 15 Gln Gln His Ser Gln Ala Leu
Pro Gly Gly Phe Asn Ala Pro Phe Asp 20 25 30 Val Gly Ile Lys Leu
Ser Gly Val Gln Tyr Gln Gln His Ser Gln Ala 35 40 45 Leu Pro Gly
Gly 50 12766PRTArtificial SequenceSynthetic Peptide 127Arg Ser Leu
Gln Asp Thr Glu Glu Lys Ser Arg Ser Phe Ser Ala Ser 1 5 10 15 Gln
Ala Asp Pro Leu Ser Asp Pro Asp Gln Met Asn Glu Asp Pro Gly 20 25
30 Gly Arg Ser Leu Gln Asp Thr Glu Glu Lys Ser Arg Ser Phe Ser Ala
35 40 45 Ser Gln Ala Asp Pro Leu Ser Asp Pro Asp Gln Met Asn Glu
Asp Pro 50 55 60 Gly Gly 65 12872PRTArtificial SequenceSynthetic
Peptide 128Thr Arg Ser Ala Trp Leu Asp Ser Gly Val Thr Gly Ser Gly
Leu Glu 1 5 10 15 Gly Asp His Leu Ser Asp Thr Ser Thr Thr Ser Leu
Glu Leu Asp Ser 20 25 30 Arg Pro Gly Gly Thr Arg Ser Ala Trp Leu
Asp Ser Gly Val Thr Gly 35 40 45 Ser Gly Leu Glu Gly Asp His Leu
Ser Asp Thr Ser Thr Thr Ser Leu 50 55 60 Glu Leu Asp Ser Arg Pro
Gly Gly 65 70 12962PRTArtificial SequenceSynthetic Peptide 129Gly
Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 5 10
15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg Pro Gly Gly Gly
20 25 30 Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg
Lys Glu 35 40 45 Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg Pro
Gly Gly 50 55 60 1306PRTArtificial SequenceSynthetic Peptide 130Gly
Val Gly Pro Ala Val 1 5 1318PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(1)Xaa can be any naturally occurring amino
acidMISC_FEATURE(3)..(3)Xaa is 0 to 4 amino acids that are not P or
Gmisc_feature(5)..(8)Xaa can be any naturally occurring amino acid
131Xaa Pro Xaa Gly Xaa Xaa Xaa Xaa 1 5 1326PRTArtificial
SequenceSynthetic Peptide 132Ala Pro Gly Val Gly Pro 1 5
1336PRTArtificial SequenceSynthetic Peptide 133Gly Arg Gly Asp Ser
Pro 1 5 1347PRTArtificial SequenceSynthetic
Peptidemisc_feature(1)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acid 134Xaa Xaa Pro Xaa Xaa Gly Xaa 1 5 1355PRTArtificial
SequenceSynthetic PeptideMISC_FEATURE(4)..(4)X is not P 135Val Pro
Gly Xaa Gly 1 5 1367PRTArtificial SequenceSynthetic
Peptidemisc_feature(3)..(3)Xaa is 0 to 4 amino acids, each of which
is independently any naturually occurring amino acid 136Arg Pro Xaa
Gly Tyr Asp Ser 1 5 1378PRTArtificial SequenceSynthetic
Peptidemisc_feature(3)..(3)Xaa is 0 to 4 amino acids, each of which
is independently any naturually occurring amino acid 137Val Pro Xaa
Ser Arg Asn Gly Gly 1 5
* * * * *