U.S. patent application number 13/837590 was filed with the patent office on 2016-09-01 for site specifically incorporated initiator for growth of polymers from proteins.
This patent application is currently assigned to FRANKLIN AND MARSHALL COLLEGE. The applicant listed for this patent is Franklin and Marshall College. Invention is credited to Saadyah Averick, Krzysztof Matyjaszewski, Ryan A. Mehl.
Application Number | 20160251467 13/837590 |
Document ID | / |
Family ID | 49778521 |
Filed Date | 2016-09-01 |
United States Patent
Application |
20160251467 |
Kind Code |
A9 |
Mehl; Ryan A. ; et
al. |
September 1, 2016 |
SITE SPECIFICALLY INCORPORATED INITIATOR FOR GROWTH OF POLYMERS
FROM PROTEINS
Abstract
The present invention is directed towards a protein-polymer
composition having a protein with a site-specifically incorporated
unnatural amino acid initiator and a covalently attached
polymer.
Inventors: |
Mehl; Ryan A.; (Corvalli,
OR) ; Matyjaszewski; Krzysztof; (Pittsburgh, PA)
; Averick; Saadyah; (Pittsburgh, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Franklin and Marshall College |
Lancaster |
PA |
US |
|
|
Assignee: |
FRANKLIN AND MARSHALL
COLLEGE
Lancaster
PA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140004591 A1 |
January 2, 2014 |
|
|
Family ID: |
49778521 |
Appl. No.: |
13/837590 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13788710 |
Mar 7, 2013 |
8816001 |
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13837590 |
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PCT/US2011/051043 |
Sep 9, 2011 |
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13788710 |
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61613178 |
Mar 20, 2012 |
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Current U.S.
Class: |
435/188 |
Current CPC
Class: |
A61K 47/61 20170801;
C08F 220/26 20130101; C12N 9/96 20130101 |
International
Class: |
C08F 220/26 20060101
C08F220/26; C12N 9/96 20060101 C12N009/96 |
Goverment Interests
GOVERNMENTAL INTEREST
[0003] Some of the work involved in the development described in
the invention described in this patent application was partially
funded by the National Science Foundation grant DMR-09-69301.
Claims
1. A protein-polymer composition comprising: a first protein with a
site-specifically incorporated unnatural amino acid having a
covalently attached polymer.
2. The protein-polymer composition of claim 1, wherein the
unnatural amino acid is site specifically incorporated into the
protein one to five times.
3. The protein-polymer composition of claim 1, wherein the
incorporated unnatural amino acid is an initiator for a controlled
radical polymerization reaction.
4. The protein-polymer composition of claim 3, wherein the
incorporated unnatural amino acid is an initiator for atom transfer
radical polymerization.
5. The protein-polymer composition of claim 1, wherein the
unnatural amino acid is represented by formula 2: ##STR00006##
wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N.sub.3,
alkoxyamine, or a thiocarbonyl thio moiety; A is O, S, or NR,
wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl; and n is 0, 1, 2, or 3; or a salt thereof.
6. The protein-polymer composition of claim 5, wherein the
unnatural amino acid is represented by formula 1: ##STR00007##
wherein X is a F, Cl, Br, I, or --N.sub.3, or a salt thereof.
7. The protein-polymer composition of claim 6, wherein X is
--N.sub.3, or a salt thereof.
8. The protein-polymer composition of claim 6, wherein X is Br or
Cl, or a salt thereof.
9. The protein-polymer composition of claim 1, wherein the polymer
comprises an incorporated cross-linking moiety.
10. The protein-polymer composition of claim 9, wherein the
cross-linking moiety comprises a polymerizable group.
11. The protein-polymer composition of claim 1, wherein the polymer
is degradable.
12. The protein-polymer composition of claim 1, wherein the polymer
comprises repeating units.
13. The protein-polymer composition of claim 12, wherein the
repeating units are selected from the group comprising
methacrylates, acrylates, acrylamides, styrenics, and
acrylamide-styrenics, or combinations thereof.
14. The protein-polymer composition of claim 1, wherein the polymer
comprises a copolymer.
15. The protein-polymer composition of claim 14, wherein the
copolymer comprises repeating units.
16. The protein-polymer composition of claim 15, wherein the
repeating units are selected from the group comprising
methacrylates, acrylates, acrylamides, styrenics, and
acrylamide-styrenics, or combinations thereof.
17. The protein-polymer composition of claim 1, comprising a second
protein with a site-specifically incorporated unnatural amino acid
having a covalently attached polymer.
18. A method for preparing a protein-polymer composition comprising
the steps of: providing a first protein containing a site
specifically incorporated unnatural amino acid initiator, a
polymerization catalyst precursor, and an organic solvent to an
aqueous solution to form an emulsion; providing a first radically
polymerizable monomer to the emulsion; and providing a catalyst
precursor reducing agent to the emulsion under conditions suitable
to initiate the controlled radical polymerization.
19. The method of claim 18, wherein the controlled radical
polymerization is an atom transfer radical polymerization.
20. The method of claim 18, wherein the polymerization catalyst
precursor is CuX' and a transition metal ligand species, wherein X'
is --Cl.sub.2 or --Br.sub.2.
21. The method of claim 18, wherein the reducing agent is ascorbic
acid or a salt thereof.
22. The method of claim 18, further comprising the step of: adding
a cross-linking agent to the emulsion.
23. The method of claim 22, wherein the cross-linking agent
comprises methacrylates, acrylates, acrylamides, styrenics, or
acrylamide-styrenics, or combinations thereof.
24. The method of claim 18, further comprising the step of: adding
a coinitiator to the emulsion.
25. The method of claim 25, wherein the coinitiator is
polyethyleneglycolisobutyryl bromide.
26. The method of claim 18, wherein the unnatural amino acid
initiator is represented by formula 2: ##STR00008## wherein R1 and
R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl,
aryl, or heteroaryl; X is F, Cl, Br, I, N.sub.3, alkoxyamine, or a
thiocarbonyl thio moiety; A is O, S, or NR, wherein R is H, C1-C8
alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is
0, 1, 2, or 3; or a salt thereof.
27. The method of claim 26, wherein the unnatural amino acid is
represented by formula 1: ##STR00009## wherein X is a F, Cl, Br, I,
or --N.sub.3, or a salt thereof.
28. The method of claim 27, wherein X is --N.sub.3, or a salt
thereof.
29. The method of claim 27, wherein X is Br or Cl, or a salt
thereof.
30. The method of claim 18, further comprising the step of: adding
a copolymer to the emulsion.
31. The method of claim 18, wherein the radically polymerizable
monomer is added to the emulsion continuously or in stages during
the polymerization process.
32. The method of claim 22, further comprising the step of: adding
a coinitiator to the emulsion.
33. The method of claim 18, further comprising the step of: adding
a second protein containing a site specifically incorporated
unnatural amino acid initiator to the emulsion.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date under
35 U.S.C. .sctn.119(a)-(d) of U.S. Provisional Patent Application
No. 61/613,178, filed Mar. 20, 2012.
INCORPORATION BY REFERENCE
[0002] Provisional application 61/381,757 filed on Sep. 10, 2010 is
hereby incorporated by reference. PCT application PCT/US2011/51043
filed on Sep. 9, 2011 is also hereby incorporated by reference.
FIELD OF THE INVENTION
[0004] The present invention is directed towards a protein-polymer
composition having a site-specifically incorporated unnatural amino
acid initiator and a covalently attached polymer, and a general
method for producing the composition through controlled radical
polymerization.
BACKGROUND
[0005] Protein-polymer hybrids have revolutionized the treatment of
disease [Chemical Reviews, 2009, 109, 5402-5436; Nat Rev Drug
Discov, 2003, 2, 347-360] and biocatalytic processes. [J. Am. Chem.
Soc., 2006, 128, 11008-11009]. Protein-polymer hybrids typically
comprise linear or branched polymers "grafted to" or "grafted from"
accessable sites within the desired protein. These protein-polymer
hybrids have already shown an impressive range of altered or
improved properties. From a therapeutic perspective, the advantages
of protein-polymer hybrids over native proteins include increased
in vivo stability, minimized immune recognition due to steric
effects, enhanced in vivo circulation, and improved therapeutic
effects. Protein-polymer hybrids have also shown an increased
solubility in non-aqueous media, which have expanded the utility of
enzymatic biocatalytic processes into the realm of organic
synthesis. [Biomacromolecules, 2009, 10, 1612-1618; Biotechnology
Progress, 1994, 10, 398-402]
[0006] Recently, the concept of protein-polymer nanogel hybrids has
been introduced in order to overcome some of the long-term
stability issues associated with protein-polymer hybrids. [J. Am.
Chem. Soc. 2006, 128, 11008-11009; J. Phys. Chem. B, 2008, 112,
14319-14324; J. Biotechnology 2007, 128, 597-605.] Some of these
issues include organic solvent solubility and deactivation of
traditional protein-polymer hybrids under harsh conditions. Both of
these characteristics are extremely important for expanding the
catalytic potential of enzymatic systems. Encapsulation of proteins
into nanogel matrices have demonstrated superior temperature and
organic solvent stability for several systems, such as carbonic
anhydrase, lipase, and horseradish peroxidase among others.
[Biomacromolecules, 2007, 8, 560-565 and 2009, 10, 1612-1618; J.
Am. Chem. Soc. 2006, 128, 11008-11009; Angew. Chem., Int. Ed, 2008,
47, 6263-6266]
[0007] Traditionally, protein-polymer hybrids are synthesized in a
two-step process. The proteins are first functionalized with
N-hydroxysuccinimide-acrylate and then copolymerized with an
acrylamide and a crosslinker using REDOX initiated free radical
polymerization. However, this process produces uncontrolled,
non-specific acrylate functionalization of the protein, and often
leads to batch-to-batch variability of protein activity. This
variability often originates from non-specific modification of
lysine residues by the acrylate chemistry, resulting in
deactivation of active sites and protein denaturing. [Nat Rev Drug
Discov, 2003, 2, 214-221] Additionally, the polymers accessible
through REDOX initiated free radical polymerizations are limited by
a number of factors, including monomer selection, particle size,
protein loading, and potential for controlled release
properties.
[0008] More recently, protein-polymer hybrids have been prepared
using controlled radical polymerization techniques (see Wang et
al., Am. Chem. Soc. 1995, 117, 5614; Matyjaszewski & Xia, Chem.
Rev. 2001, 101, 2921 ("Xie"); Matyjaszewski &Tsarevsky, Nature
Chem. 2009, 1, 276) which allow unprecedented control over polymer
dimensions (molecular weight), uniformity (polydispersity),
topology (geometry), composition and chemical functionality.
[Matyjaszewski, K., Ed, Controlled Radical Polymerization; ACS:
Washington, D.C., 1998; ACS Symposium Series 685. Matyjaszewski,
K., Ed.; Controlled/living Radical Polymerization. Progress in
ATRP, NMP, and RAFT; ACS: Washington, D.C., 2000; ACS Symposium
Series 768; Matyjaszewski, K., Davis, T. P., Eds. Handbook of
Radical Polymerization; Wiley: Hoboken, 2002; Qiu, J.; Charleux,
B.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083; Davis, K.
A.; Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1.]
[0009] While controlled radical polymerization techniques permit
greater control over the polymer's composition, there is still a
need for methods to attach those polymers to site-specific
locations on a protein. Thus far, methods for site-specific
incorporation of polymerization initiators into proteins have been
limited to the N-terminal position or specific natural amino-acid
directed linkages. Both of these suffer from challenging
purification of intermediates and/or the inability to efficiently
control the number or location of potential polymer connections,
both of which can compromise the structural integrity of the
modified protein.
[0010] While the many experiments conducted using in situ
functionalized natural amino acids on proteins have illustrated the
potential immense impact of well-defined protein-polymer hybrids,
their application is limited by technical shortcomings, and there
is a need to develop protein polymer hybrids where a desired
polymer can be attached at a site-specific location on the protein.
[See Broyer et al., J. Am. Chem. Soc. 2008, 130, 1041]
SUMMARY
[0011] In view of the above-mentioned need, a protein-polymer
composition is provided. The protein-polymer composition includes a
protein with a site-specifically incorporated unnatural amino acid
with a covalently attached polymer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Embodiments of the invention will be understood by reference
to the following figures, wherein:
[0013] FIG. 1: Shows the emission spectra for GFP-wt and
GFP-1-p(OEO300MA) .about.510 nm
[0014] FIG. 2: Catalase nanogel synthetic scheme
[0015] FIG. 3: A) Dynamic light scattering of GFP-1 peak size
.about.240 nm. B) confocal microscopy of GFP-1.
[0016] FIG. 4: Fast Protein Liquid Chromatography ("FPLC") of GFP-1
reaction, GFP-1 (0 min), GFP-1-p(OEO300MA) (180 min).
[0017] FIG. 5: Lower Critical Solution Temperature ("LCST")
behavior of GFP-1-p (OEO300MA)
DETAILED DESCRIPTION OF THE EMBODIMENT(S)
[0018] One aspect of the invention is a protein-polymer composition
having a protein with a site-specifically incorporated unnatural
amino acid with a covalently attached polymer.
[0019] The general method of preparing a protein with a
site-specifically incorporated unnatural amino acid is disclosed by
Mehl et al., PCT/US2011/57043, and is incorporated herein by
reference.
[0020] A "protein" (or portion thereof) is understood to include
native proteins, as well as proteins that have one or more
site-specifically incorporated unnatural amino acids further
comprising an initiator for a CRP. No attempt is made to identify
the hundreds of thousands of known proteins, any of which may be
modified to include one or more unnatural amino acid initiators,
e.g., by tailoring any available mutation methods to include one or
more appropriate selector codon in a relevant translation system.
Common sequence repositories for known proteins include GenBank,
EMBL, DDBJ, and the NCBI, among others. Typically, the proteins
are, e.g., at least 60%, at least 70%, at least 75%, at least 80%,
at least 90%, at least 95%, or at least 99% or more identical to
any available protein (e.g., a therapeutic protein, a diagnostic
protein, an industrial enzyme, or portion thereof, and the like),
and they can comprise one or more unnatural amino acid initiators.
Essentially any protein of interest can be modified to include an
initiator comprising an unnatural amino acid initiator.
[0021] Proteins are also understood to include enzymes (e.g.,
therapeutic, diagnostic, or industrial enzymes), or portions
thereof with at least one or more unnatural amino acid initiators
are also provided by the invention. Examples of enzymes include,
but are not limited to, e.g., amidases, amino acid racemases,
acylases, dehalogenases, dioxygenases, diarylpropane peroxidases,
epimerases, epoxide hydrolases, esterases, isomerases, kinases,
glucose isomerases, alycosidases, glycosyl transferases,
haloperoxidases, monooxygenases (e.g., p450s), lipases, lignin
peroxidases, nitrile hydratases, nitrilases, proteases,
phosphatases, subtilisins, transaminases, and nucleases.
[0022] The term "selector codon" refers to a codon recognized by
the O-tRNA in the translation process and not typically recognized
by an endogenous ERNA. The O-tRNA anticodon loop recognizes the
selector codon on the mRNA and incorporates its amino acid, e.g.,
an initiator amino acid, at this site in the polypeptide. Selector
codons can include, e.g., nonsense codons, such as stop codons
(e.g., amber, ochre, and opal codons), four or more base codons,
rare codons, codons derived from natural or unnatural base pairs,
or the like.
[0023] The term "translation system" refers to the components that
incorporate an amino acid into a growing polypeptide chain
(protein). Components of a translation system can include, e.g.,
ribosomes, tRNAs, synthetases, mRNA, and the like. Typical
translation systems include cells, such as bacterial cells (e.g.,
Escherichia coli), archeaebacterial cells, eukaryotic cells (e.g.,
yeast cells, mammalian cells, plant cells, insect cells), or the
like. Alternatively, the translation system comprises an in vitro
translation system, e.g., a translation extract including a
cellular extract. The O-tRNA or the O-RSs of the invention can be
added to or be part of an in vitro or in vivo translation system,
e.g., in an eukaryotic cell, e.g., a bacterium (such as E. coli),
or in a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a
plant cell, an algae cell, a fungus cell, an insect cell, or the
like. The translation system can also be a cell-free system, e.g.,
any of a variety of commercially available in vitro
transcription/translation systems in combination with an
O-tRNA/O-RS pair and an initiator amino acid as described
herein.
[0024] The translation system may optionally include multiple
O-tRNA/O-RS pairs, which allow incorporation of more than one
unnatural amino acid, e.g., an initiator amino acid and another
unnatural amino acid. For example, the cell can further include an
additional different O-tRNA/O-RS pair and a second unnatural amino
acid, where this additional O-tRNA recognizes a second selector
codon and this additional O-RS preferentially aminoacylates the
O-tRNA with the second unnatural amino acid. For example, a cell
that includes an O-tRNA/O-RS pair (where the O-tRNA recognizes,
e.g., an amber selector codon) can further comprise a second
orthogonal pair, where the second O-tRNA recognizes a different
selector codon (e.g., an opal codon, four-base codon, or the like).
Desirably, the different orthogonal pairs are derived from
different sources, which can facilitate recognition of different
selector codons.
[0025] An "unnatural amino acid" is, in this case a molecule
containina a primary amine functionality and carboxylic acid
functionality that can be incorporated into a protein primary
sequence with a transferable atom or group that is completely
incorporated into the final product.
[0026] In an embodiment, the unnatural amino acid is
site-specifically incorporated into the protein one to five times.
In another exemplary embodiment, the unnatural amino acid is
site-specifically incorporated into a protein one to three times.
In yet another exemplary embodiment, the unnatural amino acid is
site-specifically incorporated into a protein one to two times.
Exemplary examples include GFP-1, discussed below, wherein a single
unnatural amino acid with a covalently attached polymer was
incorporated without loss of fluorescence. FIG. 1. In another
exemplary example, incorporation of Catalase-NG with four
incorporated initiation sites into a nanogel was accomplished while
retaining the ability of Catalase to reduce hydrogen peroxide. FIG.
2 and Image 1.
[0027] As used herein, the term "nanogel" refers to a polymer
network dispersion capable of absorbing a fluid and retaining at
least a portion of the fluid to form a swollen polymer particle. A
nanogel can have many sizes, and these sizes are indicative of the
nanogel in solvent swollen form.
[0028] In an exemplary embodiment, the site specifically
incorporated unnatural amino acid is an initiator for a controlled
radical polymerization reaction ("CRP"). CRP reactions include, but
are not limited to, atom transfer radical polymerization ("ATRP"),
nitroxide mediated polymerization ("NMP"), and reversible addition
fragmentation transfer ("RAFT") systems. CRP reactions allow
unprecedented control over polymer dimensions (molecular weight),
uniformity (polydispersity), topology (geometry), composition and
functionality. [Matyjaszewski, K., Davis, T. P., Eds. Handbook of
Radical Polymerization; Wiley; Hoboken, 2002; Qiu, J.; Charleux,
B.; Matyjaszewski, K. Prog. Polym. Sci, 2001, 26, 2083; Davis, K.
A.; Matyjaszewski, K. Adv. Polym. Sci, 2002, 159, 1.]
[0029] Matyjaszewski and coworkers disclosed the fundamental four
component Atom Transfer Radical Polymerization (ATRP) process
comprising the addition, or in situ formation, of an initiator, in
this case a molecule with a transferable atom or group that is
completely incorporated into the final product, a transition metal
and a ligand that form, a partially soluble transition metal
complex that participates in a reversible redox reaction with the
added initiator or a dormant polymer to form the active species to
copolymerize radically polymerizable monomers, and a number of
improvements to the basic ATRP process, in a number of patents and
patent applications: U.S. Pat. Nos. 5,763,546; 5,807,937;
5,789,487; 5,945,491; 6,111,022; 6,121,371; 6,124,411; 6,162,882;
6,624,262; 6,407,187; 6,512,060; 6,538,091; 6,541,580; 6,624,262;
6,627,314; 6,759,491; 6,790,919; 6,887,962; 7,019,082; 7,049,373;
7,064,166; 7,125,938; 7,157,530; 7,332,550 and U.S. patent
application Ser. Nos. 09/534,827; PCT/US04/09905; PCT/US05/007264;
PCT/US05/007265; PCT/US06/33152; PCT/US2006/048656 and
PCT/US08/64710, all of which are herein incorporated by reference
to provide both background and definitions for the terms used
herein. Papers include Wang et al., Am. Chem. Soc. 1995, 117, 5614;
Matyjaszewski & Xia, Chem. Rev. 2001, 101, 2921; Matyjaszewski
& Tsarevsky, Nature Chem. 2009, 1, 276.
[0030] In an exemplary embodiment, the unnatural amino acid is an
initiator for ATRP, therefore allowing for monomers and
cross-linkers to be incorporated in a predictable, controlled, and
programmed manner to yield polymer chains of essentially equal
length, as defined by the ratio of consumed monomer to the added
initiator. Moreover, the functionality present on the introduced
initiator can be preserved, including both the .alpha.- and
.omega.-chain end functionality on the formed polymer segment. The
polymers synthesized using ATRP also may allow many functional
groups, such as hydroxyl, amino, amido, esters, carboxylic acid, to
be incorporated into a copolymer for use in post-polymerization
modifications, including covalent linking of biomolecules for drug
delivery. As disclosed below, this enables formation of
protein-polymer hybrids between synthetic polymers and
biomolecules, and provides delivery systems with customizable and
tunable polymer structures for many applications, including but not
limited to precise targeted delivery of biologically active
molecules.
[0031] An "initiator" is understood to mean a chemical species with
a transferable atom that is capable of interacting with a
transition metal and a ligand to form a partially soluble
transition metal complex that participates in a reversible redox
reaction with the added initiator or a dormant polymer to form the
active species to copolymerize radically polymerizable
monomers.
[0032] An exemplary embodiment, the unnatural amino acid is
represented by formula 2:
##STR00001##
wherein R1 and R2 are independently H, C1-C8 alkyl, cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl; X is F, Cl, Br, I, N.sub.3,
alkoxyamine, or a thiocarbonyl thio moiety; A is O, S, or NR,
wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl; and as is 0, 1, 2, or 3; or a salt thereof.
[0033] In another exemplary embodiment, the unnatural amino acid is
represented by formula 1:
##STR00002##
wherein X is F, Cl, Br, I, or --N.sub.3, or a salt thereof. In
another embodiment, the unnatural amino acid is represented by
formula 1, wherein X is Br or Cl, or a salt thereof. In yet another
embodiment, the unnatural amino acid is represented by formula 1,
wherein X is --N.sub.3 or a salt thereof.
[0034] In another embodiment, the protein-polymer composition has a
polymer with repeating units from a monomer class including
methacrylates, acrylates, acrylamides, styrenics, or
acrylamide-styrenics, or combinations thereof.
[0035] Another aspect of the invention is that the polymer in the
protein-polymer composition can include a copolymer that has
repeating units from a monomer class including methacrylates,
acrylates, acrylamides, styrenics, or acrylamide-styrenics, or
combinations thereof.
[0036] In yet another embodiment, the polymer may be copolymerized
with difunctional monomers.
[0037] In yet another embodiment, when the polymer is copolymerized
with difunctional monomers, and the protein is incorporated into a
hyperbranched structure or a nanogel.
[0038] In yet another embodiment, the polymer employed in the
protein-polymer composition can be degradable. In one non-limiting
exemplary embodiment, a single linkage point between the protein
and the polymer network can allow the protein to be efficiently
released from the conjugated polymer by cleaving a degradable link,
e.g. disulfide or acetal, as each protein is attached to the
network through only one link/chain, thereby making these
protein-polymer conjugates better suited for controlled release
applications. In another non-limiting exemplary embodiment,
multiple linkage points between the protein and the polymer network
can provide additional control over efficiently releasing the
conjugated polymer by cleaving the degradable linkages.
[0039] In an exemplary embodiment, an injectable protein-nanogel is
prepared when copolymerization is conducted in an emulsion. (Scheme
1)
##STR00003##
[0040] In another embodiment, a protein-polymer nanogel hybrid is
provided having a protein linked to one primary polymer chain at a
precise region of the protein, thereby providing greater scaffold
structural integrity whilst still forming well defined particles
due to the emulsion process employed for the synthesis of the
nanogel.
[0041] In additional embodiments, a targeted protein-polymer
nanogel system is prepared using programmable behaviors of
thermo-responsive or a pH sensitive composite structure. For
example, as an exemplary embodiment, hydroxyethylmethacrylate
(HEMA) can be chosen as a monomer for the polymerization process to
provide available functional groups for further post-polymerization
reactions and give a route for hydrogel synthesis.
[0042] In another exemplary embodiment, the polymer can be a
cross-linked polymer. In such an embodiment, a degradable
crosslinker can be used in the synthesis, which results in the
preparation of nanogels that provide for a controlled release of
proteins and bio-active molecules. Exemplary embodiments include,
but are not limited to, deliveries of polynucleotides (e.g.
oligonucleotides) and/or other therapeutic agents from the
protein-polymer hybrids. This can be important for drug delivery
applications through extended t1/2 life circulation of protein
therapeutics.
[0043] In yet another embodiment, protein-polymer hybrids can
incorporate different proteins at have synergistic activity, and
can provide multi-protein nanogels with distinct protein domains
within the nanogel. These systems have greater stability to
enzymatic degradation while providing for controlled release of
tethered bio-active agents if degradable cross-linkers are used,
and also can have increased stability in organic solvents as
compared to isolated wild-type proteins or wild-type proteins
simply entrapped in a nanogel. Scheme 1 and FIG. 2 show an
exemplary embodiment having a protein incorporated into a nanogel.
These systems are proposed to have greater stability to enzymatic
degradation, and increased potential for controlled release if
degradable cross linkers are employed.
[0044] Another aspect of the invention is a method for preparing a
protein-polymer composition having a protein with a
site-specifically incorporated unnatural amino acid covalently
attached to a polymer. The method comprises the steps of:
[0045] Providing a first protein containing a site specifically
incorporated unnatural amino acid initiator, a polymerization
catalyst precursor, and an organic solvent to an aqueous solution
to form an emulsion;
[0046] providing a first radically polymerizable monomer to the
emulsion; and
[0047] providing a catalyst precursor reducing agent is added to
the emulsion.
[0048] The process is exemplified by using a protein with a
site-specifically incorporated unnatural amino acid with a CRP
initiator functionality. Exemplary embodiments include, but are not
limited to, CRP initiators for ATRP, NMP, or RAFT. These initiators
can be introduced into nearly any protein thereby providing the
ability to advance the field of protein polymer hybrids from
non-functional proteins, e.g. bovine serum albumin, towards enzymes
or therapeutically relevant systems. Therefore, one can assay the
efficacy of the system and properly study the effects of polymer
placement using commercially available enzyme assays.
[0049] In an embodiment, the method is exemplified by an unnatural
amino acid initiator of formula 2, wherein R1 and R2 are
independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl,
or heteroaryl; X is F, Cl, Br, I, N.sub.3, alkoxyamine, or a
thiocarbonyl thio moiety; A is O, S, or NR, wherein R is H, C1-C8
alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is
0, 1, 2, or 3; or a salt thereof.
[0050] In another embodiment of the method, the unnatural amino
acid initiator is of formula 1, wherein X is a F, Cl, Br, I, or
--N.sub.3, or a salt thereof. In yet another embodiment of the
method, the unnatural amino acid initiator is of formula 1, wherein
X is --N.sub.3, or a salt thereof. In another embodiment of the
method, the unnatural amino acid initiator is of formula 1, wherein
X is Br or Cl, or a salt thereof.
[0051] In one embodiment, a coinitiator is added to the emulsion.
In an additional embodiment, polyethyleneglycolisobutyryl bromide
is a coinitiator and is added to the emulsion. In another
embodiment, a second protein containing a site-specifically
incorporated unnatural amino acid initiator is added to the
emulsion.
[0052] In another embodiment, the polymerization catalyst precursor
is a transition metal and a transition metal ligand species, that
form a partially soluble transition metal complex that participates
in a reversible redox reaction with the added initiator to form an
active species suitable for polymerization of a radically
polymerizable monomer. In an exemplary embodiment, the
polymerization catalyst precursor comprises a copper halide and a
transition metal ligand species. In another exemplary embodiment,
the copper halide is CuBr.sub.2 or CuCl.sub.2.
[0053] Exemplary examples of radically polymerizable monomers
include, but are not limited to methacrylates, acrylates,
acrylamides, styrenics, or acrylamide-styrenics, or combinations
thereof. In one embodiment, the method further comprises adding a
radically polymerizable copolymer to the emulsion. The radically
polymerizable copolymer can have repeating monomers. In an
embodiment, the copolymer monomers can include methacrylates,
acrylates, acrylamides, styrenics, or acrylamide-styrenics, or
combinations thereof. In another embodiment, the polymer is
copolymerized with difunctional monomers. In another embodiment,
the first radically polymerizable monomer is added to the emulsion
continuously or in stages during the polymerization process. In
another embodiment, the radically polymerizable copolymer is added
to the emulsion continuously or in stages during the polymerization
process.
[0054] A catalyst precursor reducing agent for CRP reactions may be
any reducing agent capable of reducing the transition metal
catalyst from a higher oxidation state to a lower oxidation state,
such as, but not limited to, ascorbic acid or salts thereof; tin
octonate, reducing sugars such as fructose, antioxidants, those
used in food preservatives such as flavonoids, beta carotene,
.alpha.-tocopherol, propyl or octyl gallate (triphenol) BHA or BHT,
or other food preservatives such as nitrites, propionic acids,
sorbates, or sulfites. In another embodiment, the catalyst
precursor reducing agent is ascorbic acid.
[0055] In another embodiment, the method further comprises the step
of adding a cross-linking reagent to the emulsion. Exemplary
example of a cross-linking reagent include, but are not limited to
methacrylates, acrylates, acrylamides, styrenics, or
acrylamide-styrenics, or combinations thereof. In another
embodiment, when a cross-linking reagent is added to the emulsion,
a coinitiator is further added to the emulsion.
##STR00004##
[0056] To demonstrate the utility of a protein-polymer hybrid
having a site-specifically incorporated unnatural amino acid
covalently attached to a polymer, a green fluorescent protein
("GFP") with the functional initiating site specifically
incorporated on sample GFP-1's surface was produced as a
non-limiting exemplary protein. See scheme 2. Using an exemplary
unnatural amino acid, 4-(2'-bromoisobutylamido)-phenylalanine,
GFP-1 was produced by replacing GFP-wt's Asp-134 through a
variation of the procedure disclosed in U.S. Pat. No. 7,776,535,
which is incorporated by reference. Specifically, the exemplary
unnatural amino acid was incorporated into a methanococcus
jannaschii (Mj) tyrosyltRNA synthetase (RS)/tRNACUA pair to
genetically encode this initiator in response to an amber codon.
Grafting from the incorporated
4-(2'-bromoisobutylamido)-phenylalanine initiator under standard
ATRP conditions with the monomer oligo(ethylene oxide) monomethyl
ether methacrylate, did not affect the green fluorescent properties
of the GFP protein, allowing the fluorescent properties to be a
measure of the influence of the conditions employed for the CRP on
the structure of the selected protein. FIG. 1 shows that the
fluorescent properties were not affected. Polymers grown from
random sites on an unmodified sample of GFP-wt resulted in a lack
of fluorescent properties by the GFP-polymer hybrid.
[0057] The exemplary GFP-1 proteins used in this work were attached
to a polymer by a single covalent linkage to the site-specifically
incorporated initiator, and the GFP-1 was incorporated into the
resulting nanogel. The nanogel incorporated GFP-1 protein was not
compromised by either the covalent unnatural amino acid-polymer
linkage or subsequent incorporation into a nanogel, which was
confirmed by the fact that the nanogels retained GFP's intrinsic
light emitting properties, FIG. 3B.
[0058] Other embodiments of the invention include the production of
functional protein-polymer hybrid materials such as enzymes and
assaying their activity under synthetically relevant conditions.
This ability to prepare functional proteins with site selected
functionality is further exemplified by preparation of a Catalase
that was bio-engineered to possess 4 ATRP initiating groups, sample
Cat-1 FIG. 2. Catalase is an enzyme that converts H.sub.2O.sub.2
into oxygen and water and it was confirmed that the enzymatic
reaction was not modified after the Catalase had undergone polymer
modification, Image 1.
[0059] One skilled in the art should appreciate that the above
steps are merely exemplary and used to enable one skilled in the
art to prepare protein-polymer compositions containing a protein
with site-specifically incorporated unnatural amino acid covalently
attached to a polymer, Additionally, those skilled in the art will
recognize, or be able to ascertain using no more than routine
experimentation, numerous equivalents to the specific procedures,
embodiments, claims, and examples described herein. Such
equivalents were considered to be within the scope of this
invention and covered by the claims appended hereto.
EXAMPLES
Example 1
Synthesis of Protein-Polymer Conjugates Via "Grafting from" an
Incorporated Functionality
[0060] The procedure used to incorporate a functional group that
can act as an initiator for an ATRP into a protein is schematically
represented in Scheme 3. The first formed ATRP initiator
4-(2'-bromoisobutyramido) phenylalanine (1) need not be asymmetric,
since the MjRS utilizes only the L form. (B).
##STR00005##
[0061] This was accomplished using an E. coli tyrosyl
tRNA/tRNA-synthesize pair vector. By using genetic engineering,
specific placement of the initiating amino acid was selected to be
expressed at the 134 position of GFP, the resulting modified
protein being GFP-1, protecting the protein's active sites and
structurally weak regions.
N-Boc-4-(2'-bromoisobutyramido)-phenylalanine
[0062] Commercially obtained N-Boc-4-aminophenylalanine (3.62 g,
0.01447 mol) was dissolved in 50 mL of dry THF, 2-bromoisobutyryl
bromide (1.757 mL, 0.01422 mol) was added dropwise over 30-60
seconds with vigorous stirring. The reaction was complete after 10
min (monitored by TLC). After approximately 20 min. the entire
reaction mixture (including newly formed precipitate) was
transferred to a separatory funnel with CHCl.sub.3, and
approximately 100 mL of H.sub.2O. The reaction mixture was
extracted with CHCl.sub.3 (3.times.50 mL). The organic phase was
washed with distilled water (2.times.50 mL) and brine (50 mL). The
organic phase was dried with MgSO.sub.4 and evaporated in vacuo to
obtain the crude product (4.55 g). The crude solid product was
recrystallized in 20-30 acetonitrile three times to purify the
product. After three recrystallizations, the desired product was
obtained in 65% yield (3.63 g). .sup.1H NMR (500 MHz, DMSO)
confirmed the structure.
4-(2'-bromoisobutyramido)phenylalanine
[0063] N-Boc-4-(2'-bromoisobutyramido)-phenylalanine (4.8 g, 0.0112
mol) was dissolved in 50 mL ethyl acetate under argon and dry 4 M
HCl in dioxane (50 mL) was subsequently added to the solution while
stirring at room temperature overnight. The reaction mixture was
then evaporated under reduced pressure to a final volume of 5-10
mL. Pentane was then added to the solution, and the precipitate was
filtered using an M type filter crucible and dried under reduced
pressure. The product was present as the HCl salt in 97% yield
(3.93 g). .sup.1H NMR (500 MHz, DMSO) confirmed the structure,
Selection of an Aminoacyl-IRNA Synthetase Specific for
4-(2'-bromoisobutyramido)phenylalanine
[0064] The library of aminoacyl-tRNA synthetases was encoded on a
kanamycin (Kn) resistant plasmid (pBK, 3000 bp) under control of
the constitutive Escherichia coli GlnRS promoter and terminator.
The aminoacyl synthetase library (3D-Lib) was randomized as
follows: Leu65, His 70, Gln155, and Ile159 were randomized to all
20 natural amino acids; Tyr32 was randomized to 15 natural amino
acids (less Trp, Phe, Tyr, Cys, and Ile); Asp158 was restricted to
Gly, Ser, or Val; Leu162 was restricted to Lys, Ser, Leu, His, and
Glu; and Phe108 and Gln109 were restricted to the pairs Tip-Met,
Ala-Asp, Ser-Lys, Arg-Glu, Arg-Pro, Ser-His, or Phe-Gln. The
library plasmid, pBK-3D-Lib, was moved between cells containing a
positive selection plasmid (pCG) and cells containing a negative
selection plasmid (pNEG).
[0065] The positive selection plasmid, pCG (10000 bp), encodes a
mutant Methanococcus jannaschii (Mj) tyrosyl-tRNACUA, an amber
codon-disrupted chloramphenicol acetyltransferase, an amber
codon-disrupted T7 RNA polymerase that drives the production of
green fluorescent protein, and the tetracycline (Tet) resistance
marker. The negative selection plasmid, pNEG (7000 hp), encodes the
mutant tyrosyl-tRNACUA, an amber codon-disrupted bamase gene under
control of an arabinose promoter and rrnC terminator, and the
ampicillin (Amp) resistance marker. pCG electrocompetent cells and
pNEG electrocompetent cells were made from DH10B cells carrying the
respective plasmids and stored in 100 .mu.L aliquots at -80.degree.
C. for future rounds of selection.
[0066] The synthetase library in pBK-3D-Lib was transformed by
electroporation into DH10B cells containing the positive selection
plasmid, pCG. The resulting pCG/pBK-3D-Lib-containing cells were
amplified in 1 L of 2.times.YT with 50 .mu.g/mL Kn and 25 .mu.g/mL
Tet with shaking at 37.degree. C. The cells were grown to
saturation, then pelleted at 5525 ref, resuspended in 30 mL of
2.times.YT and 7.5 mL of 80% glycerol, and stored at -80.degree. C.
in 1 mL aliquots for use in the first round of selections.
[0067] For the first positive selection, 2 mL of pCG/pBK-3D-Lib
cells were thawed on ice before addition to 1.2 L of room
temperature 2.times.YT media containing 50 .mu.g/mL Kn and 25
.mu.g/mL Tet. After incubation (11 h, 250 rpm, 37.degree. C.), a
200 .mu.L aliquot of these cells was plated on eleven 15 cm
GMML-agar plates containing 50 .mu.g/mL Kn, 25 .mu.g/mL Tet, and 60
.mu.g/mL, chloramphenicol (Cm). The positive selection agar medium
also contained 1 mM 1. After spreading, the surface of the plates
was allowed to dry completely before incubation (37.degree. C., 15
h). To harvest the surviving library members from the plates, 10 mL
of 2.times.YT (50 .mu.g/mL Kn, 25 .mu.g/mL Tet) was added to each
plate. Colonies were scraped from the plate using a glass spreader.
The resulting solution was incubated with shaking (60 min,
37.degree. C.) to wash cells free of agar. The cells were then
pelleted, and plasmid DNA was extracted. For the first positive
selection a Qiagen midiprep kit was used to purify the plasmid DNA.
For all other plasmid purification steps a Qiagen miniprep kit was
used to purify the plasmid DNA. The smaller pBK-3D-Lib plasmid was
separated from the larger pCG plasmid by agarose gel
electrophoresis and extracted from the gel using the Qiagen gel
extraction kit.
[0068] The purified pBK-3D-Lib was then transformed into
pNEG-containing DH10B cells. A 100 .mu.L sample of pNEG
electrocompetent cells was transformed with 50 ng of purified
pBK-3D-Lib DNA. Cells were rescued in 1 mL of SOC for 1 h
(37.degree. C., 250 rpm) and the entire 1 mL of rescue solution was
plated on three 15 cm LB plates containing 100 .mu.g/mL Amp, 50
.mu.g/mL Kn, and 0.2% L-arabinose. Cells were collected from plates
and pBK-3D-Lib plasmid DNA was isolated in the same manner as
described above for positive selections.
[0069] For the second round of positive selection, 50 ng of
purified library DNA was transformed into 100 .mu.L of pCG
competent cells. The transformants were rescued for 1.5 h in 1 mL
of SOC (37.degree. C., 250 rpm). A 50 sample of these cells was
plated on three plates prepared as described in the first positive
selection on LB agar plates.
[0070] For the second negative selection, one plate was spread with
250 .mu.L of rescued cells, and two plates were spread with 50
.mu.L of rescued cells and then incubated (12-16 h, 37.degree. C.).
For this round, the cells were plated on LB agar containing 100
.mu.g/mL Amp, 50 .mu.g/mL Kn, and 0.04% L-arabinose.
[0071] In order to evaluate the success of the selections based on
variation in synthetase efficacy (as opposed to traditional
survival/death results), the synthetases resulting from the
selection rounds were tested with the pALS plasmid. This plasmid
contains the sfGFP reporter with a TAG codon at residue 150 as well
as tyrosyl-tRNACUA. When a pBK plasmid with a functional synthetase
is transformed with the pALS plasmid and the cells are grown in the
presence of the appropriate amino acid on autoinduction agar, sfGFP
is expressed and the colonies are visibly green.
[0072] One microliter of each library resulting from the second
positive and the second negative rounds of selection was
transformed with 60 .mu.L, of pALS-containing DH10B cells. The
cells were rescued for 1 hr in 1 mL of SOC (37.degree. C., 250
rpm). A 250 .mu.L and 50 .mu.L of cells from each library were
plated on autoinducing minimal media with 25 .mu.g/mL Kn, 25
.mu.g/mL Tet, and 1 mM 1. Plates were grown at 37.degree. C. for 24
hours and then grown on the bench top, at room temperature, for an
additional 24 hours.
[0073] Autoinducing agar plates were prepared by combining the
reagents in Table 1A with an autoclaved solution of 40 g of agarose
in 400 mL water. Sterile water was added to a final volume of 500
mL. Antibiotics were added to a final concentration of 25 .mu.g/mL
Tet and 25 .mu.g/mL Kan. Nine plates were poured with 1 mM 1, and
nine plates were maintained as controls without UAA.
[0074] A total of 92 visually green colonies were selected from the
two 1 mM 1 plates and used to inoculate a 96-well plate containing
0.5 mL per well non-inducing minimal media (Table 1B, with sterile
water added to a final volume of 500 mL) with 25 .mu.g/mL Kn, 25
.mu.g/mL Tet. After 24 hours of growth (37.degree. C., 250 rpm), 5
.mu.L of these non-inducing samples were used to inoculate 96-well
plates with 0.5 mL autoinduction media (Table 1C, with sterile
water added to a final volume of 500 mL) containing 25 .mu.g/mL Kn,
25 .mu.g/mL Tet with and without 1 mM 1. Fluorescence measurements
of the cultures were collected 40 hours after inoculation using a
HORIBA Jobin Yvon FluoroMax.RTM.-4. The emission from 500 to 520 nm
(1 nm bandwidth as summed with excitation at 488 nm (1 nm
bandwidth). Samples were prepared by diluting suspended cells
directly from culture 100-fold with phosphate buffer saline
(PBS).
TABLE-US-00001 TABLE 1 Components for autoinducing and non-inducing
mediums, for final volume of 500 mL. A) Auto- B) Non- C) Auto-
induction inducing inducing medium medium plates 5% aspartate, pH
7.5 25 mL 25 mL 25 mL 10% glycerol 25 mL 25 mL 25.times. 18 amino
acid mix 20 mL 20 mL 20 mL 50.times. M 10 mL 10 mL 10 mL leucine (4
mg/mL), pH 7.5 5 mL 5 mL 5 mL 20% arabinose 1.25 mL -- 1.25 mL 1M
MgSO.sub.4 1 mL 1 mL 1 mL 40% glucose 625 .mu.L 6.25 mL 125 .mu.L
Trace metals 100 .mu.L 100 .mu.L 100 .mu.L
[0075] Fluorescence measurements of 92 synthetases with GFP clones
were conducted. Expressions of 500 .mu.L were grown for 40 hours
before dilution of suspended cells directly from culture 100-fold
with phosphate buffer saline (PBS). Fluorescence measurements were
collected using a HORIBA Jobin Yvon FluoroMax.RTM.-4. The emission
from 500 to 520 nm (1 nm bandwidth) was summed with excitation at
488 nm (1 nm bandwidth).
[0076] Fluorescence Analysis of Highest-Fluorescing Clones
[0077] Non-inducing cultures (3 mL) with 25 .mu.g/mL Kn and 25
.mu.g/mL Tet were grown to saturation (37.degree. C. with shaking
at 250 rpm) from the 20 highest-fluorescing colonies. Autoinduction
cultures (3 mL) with 25 .mu.g/mL Kn and 25 .mu.g/mL Tet were
inoculated with 30 .mu.L of non-inducing cultures and grown with
and without 1 mM 1 at 37.degree. C. with shaking at 250 rpm. After
approximately 40 hours, fluorescence was assessed. The top eight
performing clones were sequence revealing five unique members. The
top performing clone (G2) was moved from the pBK-G2 plasmid to the
pDule plasmid (PDule-BIBAF). pDule plasmid was generated by
amplifying the MjYRS gene from the pBK plasmid isolated from the
library using primers RSmovef (5'-CGCGCGCCATGGACGAATTTGAAATG-3')
and RSmover (5'-GACTCAGTCTAGGTACCCGTTTGAAACTGCAGTTATA-3'). The
amplified DNA fragments were cloned in to the respective sites on
the pDule plasmids using the incorporated NcoI and KpnI sites.
[0078] Expression and Purification of GFP-1.
[0079] DH10B E. coli cells co-transformed with the
pBad-sfGFP-134TAG vector and the machinery plasmid pDule-BIBAF were
used to inoculate 5 mL of non-inducing medium containing 100
.mu.g/mL Amp and 25 .mu.g/mL Tet. The non-inducing medium culture
was grown to saturation with shaking at 37.degree. C., and 5.0 mL
was used to inoculate 0.5 L autoinduction medium with 100 .mu.g/mL
Amp, 25 .mu.g/mL Tet, and 1 mM 1 (0.5 L of media grown in 2 L
plastic baffled flasks). After 40 hours of shaking at 37.degree.
C., cells were collected by centrifugation.
[0080] The protein was purified using BD-TALON cobalt ion-exchange
chromatography. The cell pellet was resuspended in wash buffer (50
mM sodium phosphate, 300 mM sodium chloride, pH 7) containing 1
mg/mL chicken egg white lysozyme, and sonicated 3.times.1 min while
cooled on ice. The lysate was clarified by centrifugation, applied
to 6-9 mL bed-volume resin, and bound for 30 min. Bound resin was
washed with >50 volumes wash buffer.
[0081] Protein was eluted from the bound resin with 2.5 mL aliquots
of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride,
150 mM imidazole pH 7) until the resin turned pink and the color of
the eluent the column was no longer green. The elusions
concentrations were check with a Bradford protein assay. The
protein were desalted into PBS using PD10 columns and concentrated
with 3000 MWCO centrifuge filters.
[0082] The location of incorporation of 1 at site D134 in GET
protein is indicated by the space-filling amino acid (previously
Asp) in Scheme 2. Altering the amino acid at site 134 in a flexible
loop unconnected to the chromophore does not affect the stability
or fluorescence of GFP.
[0083] MS analysis of GFP-1 confirmed the efficient high fideli
incorporation of a single unit of
4-(2'-bromoisobutyramido)phenylalanine into GFP in response to an
amber stop codon. ESI-MS-T of analysis of sfGFP shows a single
major peak at 27827.0 Da.+-.1 Da while ESI-MS-T of analysis of
GFP-1 shows a single major peak at 28024.0 Da.+-.1 Da. This shows
the expected molecular weight difference of 197 Da from native GFP
indicating a single efficient incorporation of
4-(2'-bromoisobutyramido)phenylalanine at the expected site. Each
sample did show a small peak at -131.+-.1 Da indicating minor
amounts of peptidase-based removal of N-terminal methionines and
+22 sodium adducts. No other peaks were observed that would
correlate with background incorporation of a natural amino
acid.
[0084] In summary, the evolved MjRS/tRNACUA pair in pDule-BIBAF
allows for site-specific incorporation of 1 in response to an amber
codon. The image in Scheme 3 shows expression levels of GFP-wt from
pBad-GFP-His6. Production of GFP-1 from pBad-GFP-134TAG-His6 is
dependent on 1 in the growth media: lane 3 without 1 present, lane
4 with 1 mM 1 present. The functional protein was purified by Co2+
affinity chromatography, separated by SDS-PAGE, and stained with
Coomassie.
[0085] ATRP Reactions Grafting from GFP-wt and GFP-1.
[0086] GFP-wt.
[0087] Initiator stock solution: Bpy (16.70 mg,
1.07.times.10.sup.-3 mmol) and Cu(II)Br.sub.2 (6 mg,
2.68.times.10.sup.-4 mmol) were dissolved in 10 mL of H.sub.2O; the
solution was deoxygenated with nitrogen. Cu(I)Br (3.8 mg,
2.68.times.10.sup.-4 mmol) was added to the mixture. Monomer,
OEO.sub.300MA (21 mg, 6.9.times.10.sup.-2 mmol) was added to 100
.mu.L of GFP-wt (10.2 mg, 3.4.times.10.sup.-2 mmol). This solution
was deoxygenated with nitrogen for 20 min. and then degassed
initiator solution (250 .mu.l) was added to the reaction mixture.
The zero time sample was removed and the reaction was sealed and
mixed for 3 hours then quenched by exposure to air. There was no
difference between GPC traces from first sample and final sample
indicating that no grafting from reaction occurred.
[0088] GFP-1.
[0089] Initiator stock solution: Bpy (16.70 mg,
1.07.times.10.sup.-3 mmol) and Cu(II)Br.sub.2 (6 mg,
2.68.times.10.sup.-4 mmol) were dissolved in 10 mL of H.sub.2O; the
solution was deoxygenated with nitrogen. Cu(I)Br (3.8 mg,
2.68.times.10.sup.-4 mmol) was added to the mixture. Monomer,
OEO.sub.300MA (10 mg, 3.42.times.10.sup.-2 mmol) was added to 100
of GFP-1 (6 mg, 2.14.times.10.sup.-4 mmol). This solution was
deoxygenated with nitrogen for 20 min. then degassed initiator
solution (100 .mu.L) was added to the reaction mixture. The
reaction was sealed and mixed for 3 hours then quenched by exposure
to air. The production of GFP-1-p(OEO300MA) was confirmed by FPLC
SEC analysis and SDS-PAGE. The reaction appered to have high
initiator efficiency, above 95%, as indicated by the area under the
curve for the residual GFP-1 in the GFP-p(OEO300MA) sample taken
after 180 min, FIG. 4. As can clearly be seen there is a tailing
towards low Mn region of the elutogram. The primary issue seems to
be poor deactivation since changing the monomer concentration from
.about.35% to .about.4% in aqueous solutions a tail towards low Mn
forms in the low monomer concentration case.
[0090] These protein-polymer hybrids were analyzed using dynamic
light scattering, UV-visible fluorescence spectroscopy and confocal
microscopy to confirm the successful incorporation of the protein
into the hybrid while preserving its tertiary structure, FIG.
3B.
[0091] Confirmation of Preparation of a Thermoresponsive
Protein-Polymer Hybrid.
[0092] An interesting phenomenon that is observed with the
p(OEO.sub.300MA) polymers is their LCST behavior at
.about.64.degree. C. To determine if the GFP-1-p(OEO.sub.300MA)
hybrids retained this property dynamic light scattering (DLS) was
employed to study the thermal response of the system. Setting the
temperature to 25.degree. C. and raising the temperature in
1.degree. C. steps a distinct transition from 10 nm
(GFP-1-p(OEO.sub.300MA)) to micron sized particles was clearly
observed at 64.degree. C. The GFP-1-p(OEO.sub.300MA) retained its
initial size after cooling to room temperature after 2 iterations
of LEST, FIG. 5. This exemplary thermoresponsive phenomenon of the
protein-polymer conjugate can be made more powerful when hybrid
structures with biologically relevant LCST temperature are prepared
by copolymerization procedures for potential controlled release
applications.
Example 2
Synthesis of a Protein-g-Polymer Nanogels
[0093] In order to exemplify another embodiment, cationic nanogels
were prepared by ATRP in an inverse mini-emulsion in order to
improve control over particle size and prepare functional nanogels
between 50 and 200 nm in diameter. Incorporation of a degradable
crosslinker allows bio-degradation of the crosslinkage and release
of encapsulated biomolecules and colloidal stability. Particle size
was measured using a Zetasizer Nano from Malvern Instruments.
Confocal microscopy was carried out using a Carl Zeiss LSM 510 Meta
NLO Confocor 3 Inverted Spectral Confocal Microscope using an
excitation of 488 nm. UV-vis spectroscopy was conducted on a Cary
5000 spectrophotometer and fluorescence spectra were collected on a
Cary Eclipse fluorescence spectrophotometer.
[0094] GFP-NG
[0095] To prepare the water phase of an inverse miniemulsion ATRP
Cu(II)Br.sub.2 (2.79 mg, 0.013 mmol) TPMA catalyst (3.63 mg, 0.013
mmol), 4% (w/w total solids) GFP-1(52.5 mg, 0.002 mmol),
PEO.sub.2000iBBr (50 mg, 0.025 mmol) co-initiator, oligo(ethylene
oxide).sub.300methactylate (OEO.sub.300MA) (900 mg, 3 mmol), as a
monomer, and PEO.sub.4000dimethacrylate (400 mg, 0.1 mmol) as a
crosslinking agent were dissolved in 1.46 ml 0.1M PBS buffer pH 7.4
and emulsified with a 0.05% (w/w) of span-80 in cyclohexane using
ultra sonication to form stable droplets .about.200 nm size. After
degassing, ascorbic acid was injected to reduce the Cu(II)Br.sub.2
to Cu(I)Br and initiate polymerization which was stopped after 15
hours stirring at 30.degree. C. The nanogels were purified by
precipitation by addition of the emulsion into THF followed by
extensive dialysis (50000 MWCO membrane) into water to remove
unreacted reagents. Dynamic light scattering of GFP1-NG peak showed
that the particle size .about.240 nm and confocal microscopy of
GFP1-NG showed that the nanoparticles retained their fluorescent
properties, FIG. 3B.
[0096] Catalase-NG (FIG. 2)
[0097] Catalase is an enzyme that converts H.sub.2O.sub.2 into
oxygen and water. A catalase enzyme that was been engineered with 4
ATRP initiating groups, (Cat-1), was employed as one of two
different initiators, the other was a mono-functional PEG based MI,
in an ATRP of PEO300MA and a PEG based crosslinker, PEG.sub.4000DM,
to prepare an enzyme-g-PEG conjugate nanoparticle that was
evaluated as a reducing agent.
[0098] PEO.sub.2000-iBBr (56 mg, 0.028 mmol), 238-catalse-initiator
(6.3 mg, 0.018 .mu.M 0.06 mol %, 4 weight %) OEO.sub.300MA (1008
mg, 3.36 mmol), PEG.sub.4000DM (448 mg, 0.11 mmol), CuBr.sub.2
(3.12 mg, 0.014 mmol), TPMA (4.06 mg, 0.014 mmol) were dissolved in
1.46 ml of water in a 50 ml pear shaped flask. A 0.05 w/w %
solution of Span-80 (1.46 g) in cyclohexane (29.26 g) was added to
the reaction mixture and the solution was sonicated until a stable
inverse mini-emulsion was formed. The solution was degassed and 200
.mu.L of degassed ascorbic acid in water (0.066 mg/ml) was added to
activate the catalyst by reducing a fraction of the
Cu.sup.II/complex to Cu.sup.I. After 15 hours the solid hybrid was
precipitated by addition to THF, washed twice with THF and 3 times
with water. The resulting nanogels were extensive purified using
tangential flow filtration with a 300 kDa MWCO membrane.
[0099] In both the GFP-1 and Catalase 1 examples well defined
protein nanogels were produced as measured by dynamic light
scattering (DLS), FIG. 3A. Confocal microscopy was used to study
the structure of the GFP-nanogel due to intrinsic light emitting
properties of the GFP, FIG. 3B, to determine the structural
integrity of the protein within the greater nanogel matrix. The
retention of the fluorescent properties indicate that proteins can
be subjected to grafting from reaction while retaining their shape
and biological activity.
[0100] The Catalase-nanogel could be studied by testing the
activity of this enzyme by exposing it to H.sub.2O.sub.2 to show
that it retained catalytic activity. When hydrogen peroxide was
added to an aqueous solution of the nanogels there was an immediate
evolution of oxygen; see Image 1.
[0101] The primary issue in both of the protein nanogels synthesis
was the possibility that free protein was present in the system. To
determine if any free protein is remaining a through purification
was conducted on these systems followed by a leaching assay to
determine how much protein is released into the supernatant liquid.
A protein will be considered to be covalently incorporated if after
extensive washing a constant absorbance in the nanogels is
observed.
[0102] For more conclusive proof of protein incorporation into a
nanogel the application of degradable crosslinkers can be applied.
Considering the case of Cat-NG the free Catalase can easily become
trapped into the nanogel while the Cat-1 will be covalently
attached. The use of degradable crosslinkers during the synthesis
of allows stable nanogels to be synthesized purified and then
degraded. After degradation the nanogels synthesized with Cat-1
should contain peaks for the protein-polymer hybrid while the
nanogels synthesized with Cat-wt should contain only peaks thr
Cat-wt.
Sequence CWU 1
1
2126DNAEscherichia coli 1cgcgcgccat ggacgaattt gaaatg
26237DNAEscherichia coli 2gactcagtct aggtacccgt ttgaaactgc agttata
37
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