U.S. patent application number 15/417114 was filed with the patent office on 2017-12-21 for enzyme-catalyzed synthesis of site-specific and stoichiometric biomolecule-polymer conjugates.
The applicant listed for this patent is Duke University. Invention is credited to Ashutosh Chilkoti, Yizhi Qi.
Application Number | 20170360946 15/417114 |
Document ID | / |
Family ID | 51989436 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170360946 |
Kind Code |
A1 |
Chilkoti; Ashutosh ; et
al. |
December 21, 2017 |
ENZYME-CATALYZED SYNTHESIS OF SITE-SPECIFIC AND STOICHIOMETRIC
BIOMOLECULE-POLYMER CONJUGATES
Abstract
Methods for producing polypeptide-polymer conjugates include
attachment of an initiator agent to a polypeptide specifically at
the C-terminus of the polypeptide using a sortase enzyme and in
situ polymerization of a polymer from the C-terminus. The
polypeptide-polymer conjugates may have desirable pharmacological
properties and may be used therapeutically.
Inventors: |
Chilkoti; Ashutosh; (Durham,
NC) ; Qi; Yizhi; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
51989436 |
Appl. No.: |
15/417114 |
Filed: |
January 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14894731 |
Nov 30, 2015 |
9592303 |
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PCT/US2014/040319 |
May 30, 2014 |
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15417114 |
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61828873 |
May 30, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/1077 20130101;
A61K 47/58 20170801; C08F 122/1006 20200201 |
International
Class: |
A61K 47/58 20060101
A61K047/58; C08F 122/10 20060101 C08F122/10; C07K 1/107 20060101
C07K001/107 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
R01 GM061232 awarded by the United States National Institutes of
Health, grant 5T32 GM008487 awarded by the United States National
Institutes of Health, grant R01 GM061232 awarded by the United
States National Institutes of Health, and grant R01 A146611 awarded
by the United States National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A method of making polypeptide-polymer conjugates having one or
more altered pharmacological properties from a plurality of
polypeptides having C-termini, the method comprising: (a)
contacting the plurality of polypeptides with a sortase and an
initiator agent under conditions that permit attachment of the
initiator agent to the C-terminus to form a plurality of
macroinitiators; and (b) incubating the plurality of
macroinitiators with a monomer under conditions that permit
free-radical polymerization to occur from the initiator agent to
form polypeptide-polymer conjugates, such that at least about 25%
of the polypeptides have a conjugated polymer initiated solely from
the C-terminus, wherein the polypeptide-polymer conjugates have an
altered pharmacological property selected from at least one of (i)
an in vivo half-life that is at least 25% greater compared with the
in vivo half life of the plurality of polypeptides; and (ii) an in
vivo biodistribution to a tissue, organ or disease site that is at
least 25% greater than the in vivo biodistribution of the plurality
of polypeptides.
2. The method of claim 1, wherein the plurality of polypeptides
comprise one or more peptides or protein therapeutic agents
selected from an inteferon, insulin, monoclonal antibody, blood
factor, colony stimulating factor, growth hormone, interleukin,
growth factor, therapeutic vaccine, calcitonin, tumor necrosis
factors (TNF), TNF-related apoptosis-inducing ligand (TRAIL),
glucagon-like peptide-1 (GLP-1), vasoactive intestinal peptide
(VIP), betatrophin, and enzyme.
3. The method of claim 1, wherein the monomer comprises at least
one of an acrylate, methacylate, acrylamide, and
methacrylamide.
4. The method of claim 1, wherein the polymer has side chains
comprising moieties selected from oligoethylene glycol, betaine,
carboxybetaine, sulfobetaine, phosphorylcholine, sarcosine or a
combination thereof.
5. The method of claim 1, wherein the free-radical polymerization
comprises at least one of atom transfer radical polymerization
(ATRP) and reversible addition-fragmentation chain transfer
(RAFT).
6. The method of claim 1, wherein the polypeptide comprises a
sortase recognition site, a His-tag, and elastin-responsive
polypeptide, or a combination thereof.
7. The method of claim 6, wherein the sortase recognition site
comprises LPXTG (SEQ ID NO: 3), wherein X is any amino acid.
8. The method of claim 1, wherein the sortase is Sortase A (SEQ ID
NO: 5 or SEQ ID NO: 6).
9. The method of claim 1, wherein the plurality of polypeptides and
monomer are incubated with a catalyst in step (b).
10. The method of claim 1, wherein the polypeptide-polymer
conjugates have an in vivo half-life that is at least 80% greater
than the in vivo half-life of the polypeptides.
11. The method of claim 1, wherein at least about 50% of the
polypeptides have a conjugated polymer initiated solely from the
C-terminus
12. The method of claim 1, wherein at least about 75% of the
polypeptides have a conjugated polymer initiated solely from the
C-terminus
13. The method of claim 1, wherein at least about 90% of the
polypeptides have a conjugated polymer initiated solely from the
C-terminus.
14. The method of claim 1, further comprising separating the
polypeptide-polymer conjugates formed in step b from the unreacted
macroinitiators, wherein the yield of polypeptide-polymer
conjugates is at least about 50% of the total conjugates and
macroinitiators which are separated.
15. The method of claim 14, wherein the yield of
polypeptide-polymer conjugates is at least about 75%.
16. The method of claim 14, wherein the yield of
polypeptide-polymer conjugates is at least about 85%.
17. The method of claim 14, wherein the polypeptide-polymer
conjugates are separated by chromatography, such as size-exclusion
chromatography.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/828,873, filed May 30, 2013, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0003] Proteins and peptides are becoming an important class of
therapeutic agents. Despite their high biological activity and
specificity, their therapeutic efficacy when delivered in native
form is largely hindered by numerous limitations, including short
circulation half-life, poor stability, low solubility and
immunogenicity. Frequent administration of the agent may be
required, which may increase cost, inconvenience and the risk of
adverse reactions.
[0004] Conjugating biomolecules with stealth polymers has become a
commonly used method to address these limitations.
Polypeptide-polymer conjugates are conventionally synthesized using
the "grafting to" method, in which the polymer is first
pre-synthesized, and then conjugated to the polypeptide
post-polymerizationally. Such an approach typically results in poor
yield due to steric hindrance between the biomolecule and polymer
and difficulty in product purification as a result of similar sizes
and surface properties of the reactants and products. Additionally,
conjugation is often done in a non-specific manner, by exploiting
promiscuously distributed reactive side chains or residues that are
only partially solvent accessible. As a result, large degree of
heterogeneity is often found in the product, significantly
compromising bioactivity.
SUMMARY
[0005] Methods are provided of increasing the half-life of
polypeptides, such as therapeutic polypeptides, by forming
conjugates containing a polymer and a polypeptide. In one aspect,
the methods may increase the half-life of a plurality of
polypeptides having a polymer attached at the N- or C-terminus. In
some embodiments, the polypeptides are contacted with an initiator
agent and an enzyme such as a sortase under conditions that permit
attachment of the initiator agent to at least one of the N-terminus
and the C-terminus of the polypeptides, and are incubated with a
monomer under conditions that permit polymerization to occur from
the initiator agent to form polypeptide-polymer conjugates. In some
embodiments, polymerization may occur such that at least about 25%
of the polypeptides have a conjugated polymer initiated from at
least one of the N-terminus and the C-terminus, the
polypeptide-polymer conjugates have an in vivo half-life that is at
least 50% greater than the in vivo half-life of the polypeptides or
a combination thereof.
[0006] In some embodiments, methods of making polypeptide-polymer
conjugates having one or more altered pharmacological properties
from a plurality of polypeptides having C-termini are provided. A
plurality of polypeptides are contacted with a sortase and an
initiator agent under conditions that permit attachment of the
initiator agent to the C-terminus and are incubated with a monomer
under conditions that permit free-radical polymerization to occur
from the initiator agent to form polypeptide-polymer conjugates. In
some embodiments, polymerization may occur such that at least about
25% of the polypeptides have a conjugated polymer initiated solely
from the C-terminus, and the polypeptide-polymer conjugates have an
altered pharmacological property such as an increased in vivo
half-life or an in vivo biodistribution to a tissue, organ or
disease site that is greater than the in vivo biodistribution of
the plurality of polypeptides. In some embodiments, the methods
yield, for example, at least 50%, 75% or 85% polymer-polypeptide
conjugates having a polymer solely attached at the C-terminus,
calculated as a proportion of the total polymer-polypeptide
conjugates and unreacted macroinitiators that are separated
following the polymerization reaction.
[0007] Other aspects of the invention will become apparent by
consideration of the detailed description and accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1: Synthetic route of GFP-C-poly(OEGMA). a) Recombinant
expression of ternary fusion protein GFP-srt-ELP and purification
by inverse transition cycling (ITC). b) SrtA-catalyzed
site-specific attachment of the ATRP initiator AEBMP to the
C-terminus of GFP. c) In situ ATRP of OEGMA yielding
GFP-C-poly(OEGMA).
[0009] FIG. 2: a) Photograph showing an SDS-PAGE analysis of
initiator attachment by SrtA. Lane 1: MW marker, lane 2:
GFP-srt-ELP, lane 3: SrtA, lane 4: sortase-catalyzed initiator
attachment (SCIA) reaction mixture after 5 h of reaction, lane 5:
purified GFP-C-Br macroinitiator. b) Graph depicting isotopic
distribution of GFP-C-Br C-terminal peptide
[DHMVLLEFVTAAGITHGMDELYNVDGGGSLPET-"AEBMP"].sup.3+
(DHMVLLEFVTAAGITHGMDELYNVDGGGSLPET is SEQ ID NO: 1) detected by
LC/MS-MS after trypsin digestion. c) Graph depicting SEC traces of
GFP-C-Br (rightmost peak), and three ATRP reaction (Rxn) attempts,
Rxn 1 (center-right peak), Rxn 2 (center-left peak), and Rxn 3
(leftmost peak) detected by UV absorbance at 280 nm.
[0010] FIG. 3: Drawing depicting vector map of target vector.
[0011] FIG. 4: Schematic illustration of the synthesis of ATRP
initiator
N-(2-(2-(2-(2-aminoacetamido)acetamido)acetamido)ethyl)-2-bromo-2-methylp-
ropanamide (AEBMP) (Gao, W., et al. Proc. Natl. Acad. Sci. 2009,
36, 15231).
[0012] FIG. 5: Photograph showing SDS-PAGE analysis of a)
GFP-srt-ELP purified by ITC (yield: .about.240 mg/L of
fermentation). Lane 1: marker, lane 2: E. coli lysate, lane 3:
soluble protein after one ITC cycle, lane 4, after two ITC cycles,
lane 5: after three ITC cycles, lane 6: after four ITC cycles. b)
SrtA purified by His.sub.6-tag purification (yield: .about.135 mg/L
of fermentation). Lane 1: marker, lane 2: E. coli lysate, lane 3:
first elution wash with imidazole, lane 4: second elution wash with
imidazole. c) GFP-C-Br purified by reverse His.sub.6-tag
purification. Lane 1: marker, lane 2: SCIA reaction mixture, lane
3: GFP-C-Br (without His.sub.6-tag) in first elution without
imidazole, lane 4: second elution without imidazole, lane 5: all
other His.sub.6-tagged components in first elution with imidazole,
lane 6: second elution with imidazole. d) ATRP reaction products.
Lane 1: marker, lane 2: GFP-C-Br macroinitiator before ATRP, lane
3: GFP-C-poly(OEGMA) conjugate from Rxn 1, lane 4: conjugate from
Rxn 2, lane 5: conjugate from Rxn 3, lane 6: GFP-C-Gly.sub.3
control after ATRP using Rxn 3 conditions, lane 7: GFP-C-Br
physically mixed with free poly(OEGMA) synthesized using Rxn 3
conditions. Free poly(OEGMA) does not stain due to lack of
charge.
[0013] FIG. 6: Graphs depicting a) Deconvoluted LC/ESI-MS spectra
of GFP-C-Br macroinitiator. Major peak at 28,120.4 Da agrees well
with theoretical mass of 28,123.8 Da. b) Theoretical isotopic
distribution of C-terminal peptide
[DHMVLLEFVTAAGITHGMDELYNVDGGGSLPET-"AEBMP"].sup.3+ (SEQ ID NO:
1-AEBMP) after tryptic digestion generated by Molecular Mass
Calculator software (v. 6.49, Pacific Northwest National
Laboratory, Richland Wash.).
[0014] FIG. 7: Graphs depicting SEC traces of GFP-C-Br (rightmost
peak), and conjugates from Rxn 1 (center-right peak), Rxn 2
(center-left peak), Rxn 3 (leftmost peak) detected by fluorescence
at 460 nm excitation and 507 nm emission.
[0015] FIG. 8: Graph showing fluorescence spectrum of GFP before
initiator attachment (dark squares), after initiator attachment
(light squares), and after in situ ATRP (dark circles, ATRP at Rxn
3 conditions); all samples at 20 .mu.M.
DETAILED DESCRIPTION
[0016] Polypeptide-polymer conjugates may be formed by attaching
preformed polymers with reactive end groups to targets on the
polypeptides via a variety of coupling reactions. For example,
conjugation of therapeutic proteins with polymers such as
polyethylene glycol, can prolong the serum half-life and reduce
immunogenicity of the proteins. However, the stability and
properties of these conjugates may be insufficient and difficult to
predict, because the type and frequency of attachment of the
preformed polymer may be difficult to control.
[0017] "Grafting from" techniques, or growing polymers directly
from biomolecular macroinitiators, permits more defined linkages
between the polypeptide and synthesized polymer chain. Described
herein is a technology which utilizes an enzyme to achieve
site-specific attachment of a polymerization initiator solely at
the terminus of a protein, for example the C-terminus to form a
macroinitiator, allowing for subsequent in situ growth of a stealth
polymer from the macroinitiator. The methods suitably yield
site-specific (e.g. C-terminal) and stoichiometric (one polymer
chain per protein subunit) conjugates. The enzyme-catalyzed
initiator attachment reaction can proceed with specificity, near
quantitative conversion and little or no side product. In situ
grafting of polymer from the macroinitiator can produce conjugates
with high yield and low dispersity. Both initiator attachment and
polymer grafting can be carried out in mild aqueous conditions
which do not cause denaturation of the protein. Because the product
is much larger than the reagents, the synthesized conjugates can be
purified in a single step, such as by a single run of Size
Exclusion Chromatography. Other purification techniques may be used
including, without limitation, ion exchange chromatography,
hydrophobic interaction chromatography, immobilized metal affinity
chromatography (for example if a His-tag is present, for example,
on the N-terminus of the polypeptide), range of affinity
chromatography (for example if an affinity tag is present on, for
example, the N-terminus of the polypeptide), dialysis, filtration
and ultracentrifugation using, for example, a membrane or
centrifugal filter unit with a suitable molecular weight cutoff. An
overall yield of at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, or at least about 90%
can be achieved. As the C-terminus is conserved on proteins and
peptides, the methods described herein provide a strategy for
synthesizing site-specific and stoichiometric biomolecule-polymer
conjugates with high yield.
[0018] Enzymes suitable for C-terminal attachment of an initiator
include sortases. Sortases are transpeptidases synthesized by
prokaryotic organisms which modify proteins by recognizing and
cleaving a carboxyl-terminal sorting signal. N-terminal
modification can also be achieved with Sortases, typically at a
lower efficiency than the C-terminal mechanism. The most common
substrates recognized by sortase enzymes contain the motif LPXTG
(Leu-Pro-any-Thr-Gly) (SEQ ID NO: 3), where X is any amino acid,
followed by a hydrophobic transmembrane sequence and a cluster of
basic residues, such as arginine. Cleavage occurs between the Thr
and Gly, with transient attachment of the Thr residue to the active
site Cys residue on the enzyme, followed by nucleophilic
substitution of the enzyme to complete transpeptidation. Sortases
include Sortase A, Sortase B, Sortase C and Sortase D. These four
main types can be found distributed in various strains of
gram-positive bacteria. Other Sortases include mutant sortases
produced through directed evolution which are able to recognize
alternative substrates not typically recognized in nature. Sortases
recognizing alternative substrates may be used to meet different
application needs.
[0019] Sortase A catalyzes the cleavage of the LPXTG (SEQ ID NO: 3)
motif on a target protein with the concomitant formation of an
amide linkage between a nucleophile, for example an oligoglycine
peptide, and the cleaved target protein. Suitably, the enzyme is
recombinantly expressed and purified for use in the methods
described herein. Sortase A recognizes the pentapeptide sequence
"LPXTG" (SEQ ID NO: 3; where "X" is any standard amino acid
residue) embedded in or terminally attached to a protein or
peptide, and its Cys nucleophilically attacks the amide bond
between Thr and Gly within the recognition sequence, generating a
relatively long-lived enzyme-thioacyl intermediate. To complete
transpeptidation, a second (bio)molecule with an N-terminal
nucleophilic group, typically an oligoglycine motif, attacks the
intermediate, displacing Sortase A and joining the two molecules
via a native peptide bond.
[0020] As shown in FIG. 1a, a ternary fusion protein, such as
"GFP-srt-ELP", can be recombinantly expressed to serve as the
sortase substrate. In FIG. 1, "srt" stands for the native SrtA
recognition sequence "LPETG" (SEQ ID NO: 2), and ELP (SEQ ID NO: 4)
refers to an environmentally responsive elastin-like polypeptide
(ELP) that facilitates easy purification of the ternary fusion by
inverse transition cycling (ITC), a non-chromatographic protein
purification method, and SrtA refers to Sortase A (SEQ ID NO: 5 or
6, encoded by SEQ ID NO: 11 or 12, respectively). Purification can
also be achieved for example, with a His.sub.6-tag using
immobilized metal affinity chromatography (IMAC). The
polypeptide-polymer conjugates can also be purified by other
chromatographic methods which may or may not exploit a tag or
purification moiety, such as size exclusion chromatography (SEC),
ion exchange chromatography (IEC), and hydrophobic interaction
chromatography (HIC). When ELP (SEQ ID NO: 4) is used, the
recognition sequence can be located between the protein and the ELP
(SEQ ID NO: 4), so that transpeptidation by Sortase A (SEQ ID NO: 5
or 6, encoded by SEQ ID NO: 11 or 12, respectively) not only
attaches the initiator to green fluorescent protein (GFP; SEQ ID
NO: 7) but also conveniently liberates the purification tag. As
transpeptidation relies on the presence of the enzyme, cleavage
does not begin until Sortase A is added in vitro. Very little, if
any, of the protein is thus lost in vivo before purification, hence
increasing the overall product yield.
[0021] In some embodiments the methods yield a polypeptide which
has a single polymer attached at the terminus such as the C
terminus or the N terminus. Polypeptides having a polymer attached
solely at the terminus, such as the C terminus, may constitute at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90% or at least about 95%
of the total polypeptides used in the methods and contacted with
the enzyme and initiator.
[0022] Yield of polypeptide-polymer conjugates can be high. For
example, the amount of polypeptides having a single polymer
attached at the terminus following polymerization as a proportion
of the total amount of conjugated and unconjugated polypeptides
recovered following polymerization can be at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 90%, or at least about 95%. Following the
polymerization reaction, the conjugated and unconjugated
polypeptides can be separated and the conjugates recovered by
chromatography, differential centrifugation or ultrafiltration, and
the amount of conjugated polypeptides relative to the total amount
of conjugated and unconjugated polypeptides can be calculated. For
example, a chromatograph with peaks corresponding to the conjugates
having a single polymer attached and remaining unreacted
polypeptide macroinitiator can be used to calculate the yield
efficiency.
[0023] To determine conjugation efficiency of in situ
polymerization such as ATRP from the C-terminus of a polypeptide,
for example, GFP (SEQ ID NO: 7), reaction mixtures can be analyzed
by chromatography, such as Size Exclusion Chromatography (SEC)
using either or both UV detection at 280 nm and fluorescence
detection. Exemplary chromatograms of three such reactions
(left-most peaks) are shown in FIG. 2 and FIG. 7, where in each
chromatogram, the earlier eluting peak corresponds to the
GFP-C-poly(OEGMA) conjugates and the later eluting peak around 21
min corresponds to GFP-C-Br. Area under the curve (AUC) can be used
to calculate the conjugate and unreacted protein peaks. For
example, the AUC of the GFP-C-poly(OEGMA) conjugate peak and the
residual unreacted GFP-C-Br macroinitiator peak in the chromatogram
of each polymerization reaction mixture can be computed using
software known in the art, such as EZStart software (v. 7.4,
Shimadzu). Sum of the areas of the two peaks in each chromatogram
can be regarded as 100% and the percent fraction of the conjugate
peak can be recorded as the conjugation efficiency of that
particular polymerization reaction. Values from repeated reactions
can be used to calculate the mean and standard deviation of
conjugation efficiency. For example, a calculation for
chromatograms detected by UV-vis absorbance is shown in Table 3 and
for fluorescence in Table 4.
[0024] Methods are provided for synthesizing polypeptide-polymer
conjugates, in which the polymer is formed in situ on the
polypeptide, such as at the C-terminus of the polypeptide. The
polypeptide-polymer conjugates show an increased half-life in vivo
or in serum, or other desirable pharmacological properties,
compared with polypeptides that have not had polymers formed in
situ according to methods described herein. The methods facilitate
growth of polymers that can be regulated and controlled to produce
a conjugate having particular desired features.
[0025] Methods for synthesizing polypeptide-polymer conjugates are
also provided in which a polymer is synthesized in situ from a
terminus of the polypeptide using an enzyme such as a sortase. The
C-terminus of the polypeptide, for example, is modified with an
initiator agent that facilitates polymerization from the
C-terminus. The attachment of an initiator using an enzyme such as
a sortase has the advantage that there is no requirement for fusion
to a relatively large domain, such as an intein domain, that can,
in some instances, reduce the expression of the fusion protein.
[0026] The polypeptide-polymer conjugates comprise a polypeptide to
which a polymer is attached at the C-terminus of the polypeptide.
In one embodiment, only one polymer is attached per polypeptide.
Examples of polypeptides include, but are not limited to, proteins,
and peptide sequences, such as, without limitation, peptide
sequences comprising at least about 5 amino acids, at least about
10 amino acids, at least about 20 amino acids, at least about 30
amino acids, at least about 40 amino acids, at least about 50 amino
acids, at least about 75 amino acids, at least about 100 amino
acids, at least about 150 amino acids, at least about 200 amino
acids, at least about 250 amino acids, at least about 300 amino
acids, at least about 400 amino acids, at least about 500 amino
acids, at least about 600 amino acids, at least about 700 amino
acids, at least about 800 amino acids, at least about 900 amino
acids, or at least about 1000 amino acids, or more. Examples of
proteins and polypeptides include any natural or synthetic
polypeptide that may be administered to a patient.
[0027] Examples of polypeptides include, but are not limited to,
those of interest in medicine, agriculture and other scientific and
industrial fields, particularly including therapeutic polypeptides
such as inteferons, insulin, monoclonal antibodies, blood factors,
colony stimulating factors, growth hormones, interleukins, growth
factors, therapeutic vaccines, calcitonins, tumor necrosis factors
(TNF), and enzymes. Specific examples of such therapeutic proteins
include, without limitation, enzymes utilized in enzyme replacement
therapy; hormones for promoting growth in animals, or cell growth
in cell culture; anticoagulants and active proteinaceous substances
used in various applications, for example, in biotechnology or in
medical diagnostics. Specific examples include, but are not limited
to: asparaginase; glutamase; arginase; arginine deaminase;
adenosine deaminase ribonuclease; cytosine deaminase, trypsin;
chymotrypsin, papin, epidermal growth factor (EGF), insulin-like
growth factor (IGF), transforming growth factor (TGF), nerve growth
factor (NGF), platelet-derived growth factor (PDGF), bone
morphogenic protein (BMP), fibroblast growth factor and the like;
somatostatin; somatotropin; somatropin; somatrem; calcitonin;
parathyroid hormone; colony stimulating factors (CSF); clotting
factors; tumor necrosis factors; interferons; interleukins;
gastrointestinal peptides, such as vasoactive intestinal peptide
(VIP), cholecytokinin (CCK), gastrin, secretin, and the like;
erythropoietins; growth hormone and GRF; vasopressins; octreotide;
pancreatic enzymes; dismutases such as superoxide dismutase;
thyrotropin releasing hormone (TRH); thyroid stimulating hormone;
luteinizing hormone; luteinizing hormone-releasing hormone (LHRH);
growth hormone-releasing hormone (GHRH); tissue plasminogen
activators; interleukin-1; interleukin-15; interleukin-2,
interleukin-10, GMCSF, GCSF, betatrophin, interleukin-1 receptor
antagonist (IL-1RA); glucagon-like peptide-1 (GLP-1); TNF-related
apoptosis-inducing ligand (TRAIL), glucagon-like peptide-1 (GLP-1),
vasoactive intestinal peptide (VIP), betatrophin, exenatide,
leptin, ghrelin; granulocyte monocyte colony stimulating factor
(GM-CSF); interleukin-2 (IL-2); interferons such as
interferon-.alpha.; interferon-gamma, adenosine deaminase; cytosine
deaminase, uricase; asparaginase; human growth hormone;
asparaginase; macrophage activator; chorionic gonadotropin;
heparin; atrial natriuretic peptide; hemoglobin; retroviral
vectors; relaxin; cyclosporin; oxytocin; vaccines; monoclonal
antibodies; single chain antibodies, ankyrin repeat proteins,
affibodies, and the like; and analogs and derivatives thereof.
[0028] The polymer that is grown in situ from the polypeptide
confers desirable properties to the conjugate. The term "polymer"
as used herein is intended to encompass a homopolymer,
heteropolymer, block polymer, co-polymer, ter-polymer, etc., and
blends, combinations and mixtures thereof. Examples of polymers
include, but are not limited to, functionalized polymers, such as a
polymer comprising 5-vinyltetrazole monomer units and having a
molecular weight distribution less than 2.0. The polymer may be or
contain one or more of a star block copolymer, a linear polymer, a
branched polymer, a hyperbranched polymer, a dendritic polymer, a
comb polymer, a graft polymer, a brush polymer, a bottle-brush
copolymer and a crosslinked structure, such as a block copolymer
comprising a block of 5-vinyltetrazole monomer units. Such a block
copolymer may further be capable of selective separation of closely
related chemical species such as ions, proteins or nucleic acids
via ionic bonding or complex formation.
[0029] Polymers that can be produced in situ on the polypeptide
according to the methods disclosed herein include, without
limitation, polyesters, poly(meth)acrylamides, poly(meth)acrylates,
polyethers, polystyrenes, polynorbornenes and monomers that have
unsaturated bonds. For example, amphiphilic comb polymers are
described in U.S. Patent Application Publication No. 2007/0087114
and in U.S. Pat. No. 6,207,749 to Mayes et al., the disclosure of
each of which is herein incorporated by reference in its entirety.
The amphiphilic comb-type polymers may be present in the form of
copolymers, containing a backbone formed of a hydrophobic,
water-insoluble polymer and side chains formed of short,
hydrophilic non-cell binding polymers. Examples of other polymers
include, but are not limited to, polyalkylenes such as polyethylene
and polypropylene; polychloroprene; polyvinyl ethers; such as
poly(vinyl acetate); polyvinyl halides such as poly(vinyl
chloride); polysiloxanes; polystyrenes; polyurethanes;
polyacrylates; such as poly(methyl (meth)acrylate), poly(ethyl
(meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl
(meth)acrylate), poly(tert-butyl (meth)acrylate),
poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate),
poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate),
poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl
acrylate), and poly(octadecyl acrylate); polyacrylamides such as
poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide),
poly(ethyl methacrylamide), poly(N-isopropyl acrylamide), poly(n,
iso, and tert-butyl acrylamide); and copolymers and mixtures
thereof. These polymers may include useful derivatives, including
polymers having substitutions, additions of chemical groups, for
example, alkyl groups, alkylene groups, hydroxylations, oxidations,
and other modifications routinely made by those skilled in the art.
The polymers may have side chains of betaine, carboxybetaine,
sulfobetaine, oligoethylene glycol (OEG), sarcosine or
polyethyleneglycol (PEG). For example, poly(oligoethyleneglycol
methacrylate) (poly(OEGMA)) may be used in methods of the invention
to produce polypeptide-p-OEGMA or biomolecule-poly(OEGMA)
conjugates. Poly(OEGMA) may be hydrophilic, water-soluble,
non-fouling, non-toxic and non-immunogenic due to the OEG side
chains, such that conjugating proteins or polypeptides at the N-
and/or C-termini with poly(OEGMA) can improve protein stability,
pharmacokinetics and immunogenicity.
[0030] The polypeptide-polymer conjugates may be formed by
contacting the polypeptide with an initiator and one or more
monomers under conditions that permit polymerization to occur. To
form the polymer in situ and produce a conjugate, the polypeptide
may be contacted with an initiator agent and an enzyme, such as a
sortase, under conditions that permit attachment of the initiator
agent to the polypeptide. The initiator attaches to the
polypeptide, for example, to the end of the polypeptide, such as at
one or more of the N-terminus or C-terminus of a polypeptide,
protein or combination thereof. The polypeptide and initiator may
be contacted subsequently or at least partially simultaneously with
a monomer under conditions suitable for polymerization to occur.
Accordingly, initiation sites on the polypeptide can be generated
prior to polymerization, or concurrently as polymerization occurs.
Polymerization may include, for example, atom transfer radical
polymerization (ATRP), reversible addition-fragmentation chain
transfer (RAFT) polymerization, nitroxide mediated radical
polymerization (NMP), ring-opening metathesis polymerization
(ROMP), and combinations thereof.
[0031] The methods may permit precise design of polypeptide-polymer
conjugates or protein-polymer conjugates and may provide advantages
that include a reduction or elimination of postpolymerization
modification strategies and polypeptide-polymer or protein-polymer
coupling reactions, and simplification of the purification of the
final bioconjugate from monomer, polymer and/or catalyst. The
methods may permit attachment of polymers to polypeptides in a
sample such that at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, or
at least about 95% of the polypeptides or biomolecules in the
sample have one polymer attached per biomolecule. The methods may
permit attachment of polymers to the terminus, such as the
C-terminus, of polypeptides in a sample such that at least about
10%, at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or at least about 95% of the
polypeptides or biomolecules in the sample have one polymer
attached at the C-terminus per biomolecule.
[0032] Stoichiometric attachment of one, two, three, four, five,
six, seven, eight, nine, or ten or more polymers per biomolecule
such that at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, or at
least about 95% of biomolecules in the sample have the particular
desired number of polymers attached is also permitted.
[0033] The methods may permit attachment of polymers to
polypeptides in a sample such that at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, or at least about 95% of the polypeptides have a
conjugated polymer initiated from at least one end of the
biomolecule, or from solely one end of the polypeptide, such as the
C-terminus or the N-terminus. The polypeptide-polymer conjugates
may be substantially free of attachment of polymers at sites within
the biomolecule. The polypeptide-polymer conjugates may be
substantially free of attachment of polypeptides throughout the
polymers.
[0034] For example, the methods may permit attachment of polymers
to polypeptides in a sample such that at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, or at least about 95% of the polypeptides have a
polymer attached solely or only to the C-terminus, solely or only
to the N-terminus, or solely or only to both the N and C-termini.
The polypeptide-polymer conjugates may be substantially free of
attachment of polymers at sites within the polypeptide. The
polypeptide-polymer conjugates may be substantially free of
attachment of polypeptides throughout the polymers.
[0035] A variety of monomers may be suited for use in methods of
the invention. Exemplary monomers include, but are not limited to,
lactic acid, epichlorohydrin, acrylate, methacylate, acrylamide,
methacrylamide, norbornene, and oxanorbornene. Examples of monomer
structures that may be used in ROMP, NMP, ATRP and RAFT, and other
components and techniques that may be used are described in U.S.
Patent Publication No. 20110294189, the entire disclosure of which
is herein incorporated by reference in its entirety.
[0036] The monomer may be, for example, non-biodegradable and/or
hydrophobic. The monomer may include two reactive groups, both of
which are reacted in order to form the polymer. For example, lactic
acid includes two reactive groups, a hydroxy group and a carboxy
group.
[0037] Monomers which contain one or more additional reactive
groups may be incorporated into the polymer backbone. For example,
a reactive monomer may be incorporated in the growing polymer chain
by participating in the same types of chemical reactions as the
growing polymer chain. For example, when lactide is being
polymerized using a Lewis acid catalyst, a depsipeptide (cyclic
dimer of an amino acid) can be prepared from lysine, in which the
epsilon amine group is protected, for example, with a t-boc
protecting group. The lysine is incorporated into the polymer, and
the protecting group can be removed. The resulting amine groups are
reactive with hydrophilic polymers which include leaving groups
such as tosylates, tresylates, mesylates, triflates and other
leaving groups well known to those of skill in the art.
[0038] Alternatively, the reactive monomer can include a leaving
group that can be displaced with a nucleophilic group on a
hydrophilic polymer. For example, epichlorohydrin can be used
during the polymerization step. The monomer is incorporated into
the polymer backbone, and the chloride group is present on the
backbone for subsequent reaction with nucleophiles. An example of a
hydrophilic polymer containing a nucleophilic group is a PEG with a
terminal amine group. PEG-NH.sub.2 can react with the chloride
groups on the polymer backbone to provide a desired density of
PEG-ylation on the polymer backbone. Using the chemistry described
herein, along with the general knowledge of those of skill in the
art, one can prepare polymer backbones, which include suitable
leaving groups or nucleophiles for subsequent coupling reactions
with functionalized hydrophilic polymers.
[0039] Polymers may be polymerized in situ on the biomolecule or
polypeptide at an initiation site using an initiator agent. An
initiator agent is a molecule that assists in beginning the
polymerization by interacting with the biomolecule and the monomer.
Examples of initiator agents include those compatible with ATRP
such as, without limitation,
N-(2-aminoethyl)-2-bromo-2-methylpropanamide,
N-(2-aminoethyl)-2-chloro-2-methylpropanamide,
2-bromo-N-(2-(2-hydrazinylacetamido) ethyl)-2-methylpropanamide,
2-chloro-N-(2-(2-hydrazinylacetamido) ethyl)-2-methylpropanamide.
Examples of initiator agents and systems also include those
compatible with RAFT such as, without limitation, a chain transfer
agent (CTA), ZC(.dbd.S)SR, where R can be cysteine, hydrazine,
hydroxylamine, and Z can be phenyl, alkyl, phthalimidomethyl,
coupled with traditional radical polymerization initiators
including those such as AIBN which are cleaved to initiate the
polymerization. Examples of initiators also include those
compatible with ROMP such as, without limitation, A-B, where A can
be cysteine, hydrazine, hydroxylamine, and B can be olefins.
[0040] The methods may produce protein-polymer or
polypeptide-polymer conjugates formed through site-specific
modifications of the N-terminus or C-terminus of proteins or
polypeptides with initiators such as ATRP initiators or RAFT
agents, followed by in situ ATRP and RAFT polymerization from the
initiators. The approach of modifying proteins with polymers using
the N- or C-terminus facilitates attachment of the polymer in a
defined manner because each protein usually has an N-terminus and a
C-terminus.
[0041] In a protein or polypeptide, targets for conjugation and
polymerization may include side-chains of natural amino acid (such
as lysine and cysteine) and non-canonical amino acid (such as
N6-levulinyl lysine and para-azidophenylalanine) on the surfaces of
proteins and specific interaction sites in proteins (such as
streptavidin and avidin). Particular amino acids, such as the amine
side-chain of lysine and the sulfhydryl group of cysteine may be
targeted to synthesize protein-polymer conjugates via the "grafting
from proteins" method. However, if a protein contains multiple
lysines and cysteines on their surfaces this may lead to random
modifications at multiple sites on the proteins, resulting in
ill-defined biomolecule-polymer conjugates.
[0042] Polymerization may be facilitated by the inclusion of a
catalyst solution. For example, ATRP catalyst system may include,
but are not limited to, copper halides and ligands, where ligands
can be derivatives of 2, 2'-bipyridine, other .pi.-accepting,
chelating nitrogen-based ligands such as 2-iminopyridines and some
aliphatic polyamines. RAFT catalyst system may include water
soluble radical generating compounds, such as 4,
4'-azobis(4-cyanopentanoic acid),
2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride,
2,2'-Azobis[2-(2-imidazolin-2-yl)propane]-disulfate dehydrate,
2,2'-Azobis(2-ethylpropionamidine)-dihydrochloride. ROMP catalyst
systems may include, but are not limited to, soluble Grubbs
catalysts, such as tetraethylene glycol substituted ruthenium
benzylidene, ruthenium alkylidene with friaryl phosphate ligands,
or ruthenium alkylidene with ligands with quaternary ammonium.
Other conditions used for polymerization may include, for example,
that the polymerization be carried out under low oxygen, for
example, under a noble or non-reactive gas such as argon, and/or
for a time of at least about 5 min, at least about 15 min, at least
about 60 min, and optionally no more than about 12 hr, no more than
about 24 hr, no more than about 48 hr. Polymerization may be
carried out, for example, at a temperature of at least about
5.degree. C., at least about 10.degree. C., at least about
15.degree. C., at least about 20.degree. C., at least about
30.degree. C., at least about 40.degree. C., at least about
50.degree. C., at least about 60.degree. C., at least about
70.degree. C., at least about 80.degree. C., at least about
90.degree. C. or at least about 100.degree. C.
[0043] If desired, biodegradable regions may be introduced into the
conjugates, constructed from monomers, oligomers or polymers using
linkages susceptible to biodegradation, such as, for example,
ester, peptide, anhydride, orthoester, and phosphoester bonds.
[0044] The biomolecule-polymer conjugates and polypeptide-polymer
conjugates may be used in a number of different applications. For
example, they may be used in prolonging the circulation of protein
and peptide therapeutic agents in applications that include blood
substitutes and targeting solid tumors. For example, they may be
used as therapeutic agents, imaging agents, in proteomics, as
protective coatings, in composite or smart materials, in sensors,
and in the separation or purification of biomolecules, or for
preconcentration or preprocessing of samples for assays of other
diagnostic devices. Biomolecule-polymer conjugates made as
described herein also may be useful in the treatment of diseases
and conditions, including, for example, rheumatoid arthritis,
Gaucher's disease, hyperuricemia, cancers, solid tumors, diabetes,
Alzheimer's disease, hairy cell leukemia, multiple myeloma,
venereal warts, AIDS-related Kaposi's sarcoma, chronic hepatitis B
and C, inflammatory diseases, autoimmune diseases, infectious
diseases and haemostatic disorders.
[0045] When used therapeutically, the biomolecule-polymer
conjugates and polypeptide-polymer conjugates may have properties
that result in improved targeted delivery of biomolecules to
disease sites and may thus provide enhanced diagnostic and
therapeutic efficacy of these compounds.
[0046] The biomolecule-polymer conjugates exhibit desirable
properties over non-conjugated biomolecules, polypeptides and
proteins, or over polymer conjugates formed using methods other
than those described herein. For example, biomolecule-polymer
conjugates produced as described herein may show improvement in one
or more of solubility, stability, pharmacokinetics, immunogenicity
and biodistribution or bioaccumulation at the cell, tissue, disease
site, or organ level. The improved stability of the conjugates may
manifest as an improvement in the half-life compared with a
comparable biomolecule that is not conjugated to a polymer.
[0047] The in vivo or serum half-life may be improved. In
pharmacokinetics, the half-life is calculated using a start point
when the administered pharmaceutical reaches equilibrium following
administration. The half-life is the time period in which the
pharmaceutical decreases to half the value at equilibrium. The
distribution half-life is the period where the decrease occurs due
to distribution of the pharmaceutical to the tissue reservoirs, and
is typically a steeper curve. The elimination half-life is the
period where the decrease occurs due to metabolism and elimination
of the pharmaceutical. An area-under the curve analysis can be used
to determine the in vivo half-life accounting for the decrease from
both distribution and elimination.
[0048] The improved half-life (in vivo half-life, distribution
half-life, elimination half-life or combination thereof) of the
biomolecule-polymer may be at least about 25% greater, at least
about 30% greater, at least about 40% greater, at least about 50%
greater, at least about 60% greater, at least about 70% greater, at
least about 80% greater, at least about 90% greater, at least about
100% greater, at least about 200% greater, at least 300% greater,
at least 400% greater, or at least 500% greater than the in vivo
half-life of the biomolecule when the biomolecule has not been
conjugated to a polymer according to methods of the invention.
[0049] Improved stability may also manifest as an increased shelf
life of the biomolecule, for example by reducing aggregation of the
biomolecules. For example, after storage at 4.degree. C. or
20.degree. C. for a period of about one month, about three months
or about a year, the biomolecule-polymer conjugate may show less
than about 10%, less than about 20%, less than about 30%, less than
about 40%, less than about 50%, less than about 60%, less than
about 70%, less than about 80%, or less than about 90% of the
aggregation that occurs when the biomolecule has not been
conjugated to a polymer according to methods of the invention.
[0050] The improved solubility may manifest as an improvement in
the solubility of the biomolecule-conjugate, such that the
solubility of the biomolecule-polymer conjugate is at least about
25% greater, at least about 30% greater, at least about 40%
greater, at least about 50% greater, at least about 60% greater, at
least about 70% greater, at least about 80% greater, at least about
90% greater, or at least about 100% greater than the solubility of
the biomolecule that has not been modified according to methods of
the invention. Aggregation of biomolecules, such as proteins and
polypeptides, may also be controlled or reduced by improving
solubility of the biomolecule, polypeptide or protein according to
methods of the invention
[0051] The improvement in pharmacokinetics may include an
improvement in one or more of the following: liberation of the
biomolecule-polymer conjugate when administered in a formulation,
absorption into the body, dispersion or dissemination of the
biomolecule-polymer conjugate throughout the fluids and tissues of
the body, and metabolism of parent compounds into daughter
metabolites. For example, the conjugate may effect a reduction in
metabolism of an active compound, or may stimulate metabolism of an
inactive compound to form active metabolites and a reduced rate of
excretion of an active compound from the body.
[0052] The improvement in immunogenicity may manifest as an
improvement reduction in the immune response to a biomolecule, such
that the biomolecule-polymer conjugate or polypeptide-polymer
conjugate evokes at least about a 10% reduction, at least about a
20% reduction, at least about a 30% reduction, at least about a 40%
reduction, at least about a 50% reduction, at least about a 60%
reduction, at least about a 70% reduction, or at least about a 80%
reduction in the immune response against the conjugate.
[0053] The in vivo biodistribution of the biomolecule-polymer
conjugate, such as a polypeptide-polymer conjugate, to a cell,
tissue, organ or disease site, such as a tumor or arterial plaque,
may be increased compared with the biodistribution of the
non-conjugated biomolecule. Biodistribution as used herein means
the extent to which the conjugates accumulate in a cell, tissue,
organ or disease site. For example, the biodistribution of the
biomolecule-conjugate to a cell, tissue, organ or disease site may
be at least about 10%, at least about 20%, at least about 25%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 100%, at least about 125%, at least about 150%,
at least about 200%, at least about 300%, at least about 400%, or
at least about 500% greater compared with the biomolecule not
conjugated to a polymer.
[0054] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any nonclaimed element as essential to the practice of
the invention.
[0055] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
EXAMPLES
Materials Used in Examples 1-17
[0056] All molecular biology reagents were purchased from New
England Biolabs, unless otherwise specified. All chemical reagents
were purchased from Sigma Aldrich and used as received, unless
otherwise specified.
Example 1: GFP-srt-ELP Cloning, Expression and Purification
[0057] The gene for GFP (SEQ ID NO: 7) was PCR-amplified from an
available GFP-containing pET32b(+) vector using the forward and
reverse primers:
TABLE-US-00001 GFP-F: (SEQ ID NO: 8) 5' TTCCCCTCTAGAAATAATTTTGT 3'
GFP-R: (SEQ ID NO: 9) 3'
CTACTTGACATGTTGCAGCTGCCGCCACCCCCGTCGAACGGCCTTT GGCCGCCATTCGAAACGAAC
5'
[0058] The vector map of the target vector is shown in FIG. 3 (not
to scale).
[0059] The GFP-F primer (SEQ ID NO: 8) was designed to anneal at
the RBS site immediately upstream of the target GFP sequence and
includes an Xbal site for cloning into the target vector (FIG. 3).
The GFP-R primer (SEQ ID NO: 9) was designed to anneal to the C
terminus of GFP (SEQ ID NO: 7) and includes an overhang that codes
for a Gly.sub.4Ser linker (SEQ ID NO: 10) and the `LPETG` SrtA
recognition sequence (SEQ ID NO: 2) as well as a HindIII site for
cloning into the target vector.
[0060] The GFP-srt fragment was amplified in two 50 .mu.L PCR
reactions, each containing 25 .mu.L GoTaq green master mix, 10
.mu.mol each of forward and reverse primers, 0.25 .mu.L template
and nuclease-free water in a total volume of 50 .mu.L. The PCR
reaction conditions were: 95.degree. C. for 2 min for initial
denaturation, followed by 40 cycles at 95.degree. C. for 30 s,
52.degree. C. for 30 s, and 72.degree. C. for 1 min. The resulting
`GFP-srt` PCR product was purified using a PCR purification kit and
visualized on a 1% agarose gel stained with SYBR.RTM. Safe DNA
stain. 1.5 .mu.g of the GFP product was then digested with 2 .mu.L
each of Xbal and HindIII in 1.times.NEB buffer 2 and 1.times.
bovine serum albumin (BSA) for 1.5 h at 37.degree. C. and then
purified using a PCR purification kit (QIAquick, QIAGEN).
[0061] A previously constructed pET25b(+) vector encoding a
protein-srt-His6-ELP fusion gene was used as the target vector. In
this vector, the protein-srt insert was flanked by Xbal and HindIII
restriction sites followed by codons that encode a His6-tag, a
thrombin cleavage site and an ELP with a sequence of
(VPGXG).sub.90, where X represents alanine (A), glycine (G) and
valine (V) (SEQ ID NO: 4) at 2:3:5 molar ratio. 1.5 .mu.g of this
target vector was digested with 2 .mu.L each of Xbal and HindIII in
1.times.NEB buffer 2 with 1.times.BSA for 1.5 h at 37.degree. C.,
enzymatically dephosphorylated with 1 .mu.L CIP for 15 min to 1 h
at 37.degree. C. (to prevent self-circularization of the vector),
and then purified using a PCR purification kit (QIAquick,
QIAGEN).
[0062] The `GFP-srt` PCR insert (5 .mu.L) was ligated into the
target vector (3 .mu.L) using 4 .mu.L of T4 ligase in 1.times.T4
ligase buffer and nuclease-free water in a total volume of 20
.mu.L. The ligation mixture was incubated at room temperature for 1
h, and BL21 (DE) cells were then transformed with 7 .mu.L of the
ligation mixture for 15 min in an ice-water bath, heat-shocked at
42.degree. C. for 30 s, and returned to the ice-water mixture for
another 2 min. The cells were recovered in SOC media while
horizontally shaking at 200 rpm at 37.degree. C. for 40-60 min, and
were then plated on TB agarose plates containing 100 .mu.g/mL
ampicillin (Calbiochem). Several clones were grown overnight in 3
mL TB media supplemented with 100 .mu.g/mL ampicillin, and the
plasmids were isolated by a miniprep plasmid purification kit
(Qiagen) for DNA sequence verification.
[0063] Expression and purification of the fusion protein was
carried out using modified techniques (Gao, W., et al. A. Proc.
Natl. Acad. Sci. 2010, 107, 16432; Meyer, D. E., et al. Nat.
Biotechnol. 1999, 14, 1112). Briefly, cells were cultured in
Terrific Broth (TB, Mo Bio Laboratories, Inc.) supplemented with
100 .mu.g/mL of ampicillin at 37.degree. C. Once the optical
density at 600 nm (OD600) of the culture reached 0.6, Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG, AMRESCO) was added to a
final concentration of 0.5 mM to induce overnight expression. Cells
were harvested 15 h post induction by centrifugation at 700.times.g
for 10 min and were lysed by sonication on a Misonex Ultrasonic
Liquid Processer (Qsonica, LLC.) at amplitude 85 for 3 min. Nucleic
acids non-chromatographic purification were removed from the crude
extract by addition of 1% v/v polyethyleneimine (PEI, Acros)
followed by centrifugation at 4.degree. C. at 21,000.times.g for 10
min.
[0064] The ELP tag enable of the fusion by Inverse Transition
Cycling (ITC), a non-chromatographic method we have developed for
the purification of ELP fusion proteins that takes advantage of
their inverse phase transtion behavior. After triggering the
inverse phase transition of the fusion by addition of 1M NaCl, the
aggregated proteins were collected by centrifugation at
21,000.times.g for 10 min at .about.35.degree. C. The pellet was
then resolubilized in cold PBS and the resulting solution was
centrifuged at 4.degree. C. at 21,000.times.g for 10 min to remove
any remaining insoluble material. The last two steps were repeated,
typically three or four times, until satisfactory purity was
achieved as verified by SDS-PAGE. In the final step, the protein
was resolubilized in sortase buffer (50 mM Tris-HCl, 150 mM NaCl,
10 mM CaCl.sub.2, pH 7.5) in preparation for sortase catalyzed
initiator attachment (SCIA). Protein concentration and yield were
assessed on an ND-1000 Nanodrop Spectrophotometer (Thermo
Scientific) by UV-vis absorption spectroscopy.
Example 2: Sortase Cloning, Expression and Purification
[0065] The gene for SrtA with a 59 N-terminal amino acid truncation
(previously shown to not affect its transpeptidase activity)(SEQ ID
NO: 5, encoded by SEQ ID NO: 11) and an N-terminal His6-tag in a
pET15b vector was transformed into BL21 E. coli cells. Expression
of protein and cell lysis was carried out identically as for the
GFP-srt-ELP fusion protein. The SrtA fusion protein was purified by
immobilized metal affinity chromatography (IMAC) on HisPur.TM.
cobalt spin columns (Thermo Scientific) and following the
manufacturer protocol. Briefly, the cell lysate was mixed with
equal volume of equilibration buffer (50 mM sodium phosphate, 300
mM sodium chloride, 10 mM imidazole; pH 7.4) and was loaded onto a
pre-equilibrated HisPur.TM. column. After rotating the loaded
columns at 4.degree. C. for 30 min to maximize binding, unbound
proteins were eluted by centrifugation at 700.times.g for 2 min.
Additional equilibration washes were performed until absorbance
measurement at 280 nm of the eluent reached baseline as monitored
on a ND-1000 Nanodrop Spectrophotometer. Concentration and yield at
each step were calculated from the absorbance measurements. The
bound (His).sub.6-SrtA fusion protein was eluted by centrifugation
at 700.times.g for 2 min in elution buffer (50 mM sodium phosphate,
300 mM sodium chloride, 150 mM imidazole; pH 7.4). Typically the
first two elution washes were collected and were solvent exchanged
by overnight dialysis against sortase buffer in preparation for
further use.
Example 3: ATRP Initiator Synthesis
[0066] FIG. 4 shows the pathway for the synthesis of ATRP initiator
N-(2-(2-(2-(2-aminoacetamido)acetamido)acetamido)ethyl)-2-bromo-2-methylp-
ropanamide (AEBMP) (Gao, W., et al. Proc. Natl. Acad. Sci. 2009,
36, 15231).
[0067] tert-butyl (2-(2-bromo-2-methylpropanamido)ethyl)carbamate
(1) as shown in FIG. 4 was synthesized as follows: Over a period of
15 min, 2-bromoisobutyryl bromide (3.9 mL, 31.2 mmol) was added to
a NaCl/ice cooled bath solution of N-Boc-ethylenediamine (5.01 g,
31.2 mmol) and diisopropylethylamine (6 mL, 34 mmol, 1.1 eq.) in
anhydrous dichloromethane (35 mL). After 1 h, the ice bath was
removed and the reaction was allowed to warm to room temperature
and stirring was continued for 18 h. Silica gel (.about.10 g) was
added and the mixture was concentrated to dryness under reduced
pressure on a rotary evaporator. Flash column chromatography
(RediSepRf SiO2 (80 g), 100% CH.sub.2Cl.sub.2.fwdarw.50% ethyl
acetate (EtOAc) in CH.sub.2Cl.sub.2) gave 1 as an off-white solid
(7.36 g, 75%). .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.2 (bs,
1H), 4.91 (bs, 1H), 3.33 (m, 4H), 1.93 (s, 6H), 1.43 (s, 9H).
.sup.13C NMR (CDCl.sub.3, 300 MHz): .delta. 172.9, 157.1, 80.1,
61.9, 42.0, 40.0, 32.5, 28.6. EIMS m/z: 331 ([M+Na].sup.+), 333
([M+Na].sup.+).
[0068] N-(2-aminoethyl)-2-bromo-2-methylpropanamide hydrochloride
(2) as shown in FIG. 4 was synthesized as follows: A solution of 1
(7.36 g, 23.8 mmol) in 4 M HCl in 1,4-dioxane (64 mL, 256 mmol) was
stirred at room temperature for 1 h. The reaction mixture was
concentrated to dryness on a rotary evaporator and further dried
under high vacuum using a vacuum manifold connected to a vacuum
pump, giving an off-white solid. The solid was triturated under
diethyl ether (Et.sub.2O, 3.times.100 mL) and the supernatant was
removed by careful decantation. The insoluble material was dried
under reduced pressure on a rotary evaporator giving 2 as a pale
solid (5.8 g, 99%). .sup.1H NMR (CD.sub.3OD, 300 MHz): .delta. 8.36
(bs, 1H), 3.65 (bs, 1H), 3.51 (s, 2H), 3.09 (s, 2H), 1.94 (s, 6H).
.sup.13C NMR (CD.sub.3OD, 300 MHz): .delta. 174.3, 58.9, 39.3,
37.8, 30.7. EIMS m/z: 209 ([M-Cl].sup.+), 211 ([M-Cl].sup.+).
[0069] tert-butyl
(14-bromo-14-methyl-2,5,8,13-tetraoxo-3,6,9,12-tetraazapentadecyl)carbama-
te (3) as shown in FIG. 4 was synthesized as follows:
Diisopropylethylamine (10.4 mL, 60 mmol, 2.5 eq.) was added in one
portion to an ice-bath cooled suspension of 2 (5.8 g, 23.8 mmol),
Boc-Gly-Gly-Gly-OH (6.9 g, 23.8 mmol), and EDC (6.84 g, 36 mmol,
1.5 eq.) in anhydrous CH.sub.2Cl.sub.2 (80 mL). The mixture was
stirred overnight (16 h) then diluted with CH.sub.2Cl.sub.2 (80
mL). Insoluble material was isolated by vacuum filtration and the
filter cake was washed sequentially with H.sub.2O (100 mL), cold
MeOH (3.times.20 mL), Et.sub.2O (2.times.100 mL) and dried in vacuo
giving 3 as a white powder (9.14 g, 80%).sup.1H NMR (CDCl.sub.3,
300 MHz): .delta. 4.15-4.10 (m, 4H), 3.89 (s, 2H), 3.72 (m, 4H),
1.91 (s, 6H), 1.43 (s, 9H). .sup.13C NMR (CDCl3, 300 MHz): .delta.
172.4, 168.9, 164.5, 156.8, 79.5, 52.8, 44.9, 44.4, 44.1, 38.2,
29.3, 25.2. EIMS m/z: 503 ([M+Na].sup.+), 505 ([M+Na].sup.+).
[0070]
N-(2-(2-(2-(2-aminoacetamido)acetamido)acetamido)ethyl)-2-bromo-2-m-
ethylpropanamide hydrochloride (4) as shown in FIG. 4 was
synthesized as follows: A solution of 3 (9.0 g, 18.8 mmol) in 4 M
HCl in 1,4-dioxane (80 mL, 320 mmol) was stirred at room
temperature for 1 h. The reaction mixture was diluted with
Et.sub.2O (300 mL). Insoluble material was collected and dried by
vacuum filtration, giving the product as a white powder (7.7 g,
98%). .sup.1H NMR (CD3OD, 500 MHz): .delta. 4.20 (m, 4H), 3.85 (s,
2H), 3.70 (m, 4H), 1.92 (s, 6H). (CDCl.sub.3, 300 MHz): .delta.
171.0, 169.8, 166.5, 163.8, 52.6, 43.3, 42.7, 38.8, 30.3. EIMS m/z:
380 ([MH-Cl].sup.+), 382 ([MH-Cl].sup.+).
Example 4: Sortase Catalyzed Initiator Attachment and Product
Separation
[0071] A reaction mixture consisting of GFP-srt-ELP, SrtA, and
AEBMP at a 2:1:60 ratio in sortase buffer was incubated at
37.degree. C. for 5 h. Post reaction, a reverse His-tag
purification was used to isolate the GFP-C-Br macroinitiator, by
exploiting the fact that the macroinitiator is the only species in
the mixture without a His6-tag. Equilibration and elution washes
were done as described above. The first two equilibration washes
containing the eluted GFP-C-Br were collected and solvent exchanged
by overnight dialysis against PBS in preparation for use. A control
reaction was done by replacing AEBMP with Gly.sub.3, while keeping
all other conditions the same. The resulting GFP-C-Gly.sub.3 was
used as negative control in the subsequent in situ ATRP
reaction.
Example 5: In Situ ATRP from GFP-C-Br and Conjugate
Purification
[0072] ATRP reactions were performed using conditions described
previously with minor changes. (Gao et al. Proc. Natl. Acad. Sci.
2010, 107, 16432; Gao et al. Proc. Natl. Acad. Sci. 2009). OEGMA
(MW=500) was eluted through a column packed with aluminum oxide to
remove the polymerization inhibitor. Three sets of reaction
conditions were attempted and the parameters are summarized in
Table 1.
TABLE-US-00002 TABLE 1 ATRP reaction conditions for reactions (Rxn)
1, 2, and 3. GFP-C--Br CuCl CuCl.sub.2 HMTETA OEGMA (.mu.mol/eqv)
(.mu.mol/eqv) (.mu.mol/eqv) (.mu.mol/eqv) (.mu.mol/eqv) time Rxn 1
0.2/1 5.1/25 15.0/75 25.0/125 110/550 30 m Rxn 2 0.2/1 5.1/25
11.1/55 20.0/100 220/1100 30 m Rxn 3 0.2/1 5.1/25 11.1/55 20.0/100
440/2200 2 h
[0073] Polymerization was typically carried out by first mixing
specified amounts of CuCl, CuCl.sub.2, and
1,1,2,7,10,10-hexamethyltriethylenetetramine (HMTETA) in 100 .mu.L
MilliQ water until all reagents were completely dissolved and then
topping up with 400 .mu.L PBS. A second solution was prepared by
adding OEGMA to 2 mL of 100 .mu.M GFP-C-Br in PBS. The two
solutions were degassed by bubbling separately with argon for 30
min using a Schlenk line, after which the first solution was
quickly transferred into the second solution by a cannula.
Polymerization was allowed to proceed for a specified time at room
temperature under argon and was quenched by bubbling with air. An
initial separation of the conjugate from the small MW reagents was
carried out by gel filtration on disposable PD-10 columns (GE Life
Science) before subsequent purification and characterization.
Example 6: Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) and Initiator Attachment Efficiency
[0074] Samples were prepared in Laemmli loading dye containing 5%
v/v .beta.-mercaptoethanol. After brief heating at 98.degree. C.,
the samples were loaded onto precast 4-20% Tris-HCl gels (Bio-Rad).
Gels were run at 130 V and 400 mA for 55 min in 1.times. running
buffer (25 mM Tris, 192 mM Glycine, and 0.1% SDS) on a Bio-Rad
Mini-PROTEAN gel apparatus. Gels were stained with copper
chloride.
[0075] To determine efficiency of SCIA, quantification of gel band
intensity was performed using ImageJ. This method for
quantification of the yield of initiator attachment to GFP (SEQ ID
NO: 7) is valid because we do not expect that attachment of the
initiator to GFP should alter its staining by the dye. For each
SDS-PAGE gel, bands in each lane were defined in ImageJ and
converted into intensity profile plots using a built-in function,
where each band was assigned a corresponding peak. After defining
the baseline for each peak, band intensities were computed by
calculating the area under each peak. Values were then imported
into Excel (Microsoft) for analysis.
[0076] Because the errors involved in sample loading can be
significant when the product of a sortase cleavage reaction is
normalized to a standard amount of GFP-srt-ELP loaded in a separate
lane, the yield of each SCIA reaction was calculated by internal
normalization, wherein we assume that the intensity of the products
of a sortase cleavage reaction sums to that of the parent fusion
construct it was derived from. Hence, the band intensity of the
initial amount of GFP-srt-ELP used in each reaction was determined
by summing up all of its products after reaction, namely residual
unreacted GFP-srt-ELP, cleaved ELP, and transpeptidized GFP-C-Br.
The % unreacted product is thus the band intensity of unreacted
GFP-srt-ELP divided by the sum of all products and multiplied by
100, and % transpeptidation is thus 100%-% unreacted. A very faint
band slightly above 50 kDa could also be observed upon close
inspection, which corresponds to an intermediate species of the
reaction, where SrtA is linked to the C-terminus of GFP via a
thioacyl bond. The presence of SrtA in this species makes its
staining not directly comparable to that of the other species, so
that the intensity of this bad was not incorporated into the
overall calculation of reaction yield. However, including it in the
sum of intensities and taking its percentage showed that this
intermediate only comprised <1% of the overall intensity at most
(Table 2), so that omitting it in the calculation of %
transpeptidation yield does not significantly change the
results.
[0077] Table 2 shows initiator attachment efficiency (%
transpeptidation) of SCIA determined by SDS-PAGE gel band
quantification averaged across five SCIA reactions. The intensity
of initial GFP-srt-ELP was determined by summing intensities of
unreacted GFP-srt-ELP, cleaved ELP, and transpeptidized GFP-C-Br.
The percentage unreacted was calculated as the fraction of
unreacted GFP-srt-ELP divided by sum of the three bands and
multiplying by 100. The percentage transpeptidized was calculated
as 100%-percentage unreacted. A very faint GFP-SrtA intermediate
band was also observed in all reactions, but only comprised of up
to .about.1% in all cases. Because the presence of Sortase A in the
intermediate alters its staining compared to the other three
species, making direct quantitative manipulation difficult, its
intensity was excluded from the calculation. Percentage
transpeptidation was 96.3.+-.1.5%.
TABLE-US-00003 TABLE 2 initiator attachment efficiency (%
transpeptidation) of SCIA. Intensity Unreacted Total GFP- %
Intensity GFP- Cleaved w/o SrtA Total w/ % % % srt-ELP ELP
GFP-C--Br intermediate intermediate intermediate Intermediate
Unreacted Transpeptidation SCIA #1 203.7 1726.3 1810.2 3740.2 40.6
3780.8 1.1 5.4 94.6 SCIA #2 268.3 5899.2 6970.4 13137.9 87.4
13225.3 0.7 2.0 98.0 SCIA #3 505.6 4990.1 4971.3 10466.9 101.8
10568.7 1.0 4.8 95.2 SCIA #4 207.8 3783.8 4209.0 8200.6 61.1 8261.6
0.7 2.5 97.5 SCIA #5 538.9 6955.8 7421.7 14916.4 110.6 15026.9 0.7
3.6 96.4 Mean .+-. 96.3 .+-. 1.5 Std. Dev.
Example 7: Liquid Chromatography Electrospray-Ionization Mass
Spectrometry (LC/ESI-MS)
[0078] Samples at a concentration of 5 .mu.M were first desalted by
dialyzing against MilliQ water overnight. LC/ESI-MS was performed
on an Agilent 1100 LC/MSD Quadrupole Mass Spectrometer. The
instrument was calibrated with Cytochrome C and BSA. The ESI source
was set to operate at 300.degree. C. with a nebulizer gas pressure
of 20 psi and a dry gas flow rate of 7 L/min. 1 .mu.L of sample was
separated by reverse phase chromatography on a Zorbax SB-C18 column
(Agilent) at 20%-80% acetonitrile/water gradient and a flow rate of
60 .mu.L/min. Spectra were acquired in positive ion mode over the
mass to charge range (m/z) of 400-1,600. Theoretical MW of GFP-C-Br
was calculated using Molecular Weight Calculator (v. 6.49, Pacific
Northwest National Laboratory, ncrr.pnl.gov/software).
Example 8: Nano-Flow Liquid Chromatography Electrospray Ionization
Tandem Mass Spectrometry (LC/MS-MS)
[0079] 100 .mu.L of .about.8 .mu.M sample was loaded on to a 0.5 mL
ZebaSpin desalting column (Thermo Scientific) for solvent exchange
into 50 mM ammonium bicarbonate, pH 8.0, supplemented with 0.1%
Rapigest SF surfactant (Waters Corp), by washing the loaded column
with 300 .mu.L of the solvent solution four times. The sample was
then reduced with 5 mM dithiolthreitol for 30 min at 70.degree. C.
and free sulfhydryls were alkylated with 10 mM iodoacetamide for 45
min at room temperature. Proteolytic digestion was accomplished by
the addition of 500 ng sequencing grade trypsin (Promega) directly
to the resin with incubation at 37.degree. C. for 18 h. Supernatant
was collected following a 2 min centrifugation at 1,000 rpm,
acidified to pH 2.5 with TFA and incubated at 60.degree. C. for 1 h
to hydrolyze remaining Rapigest surfactant. Insoluble hydrolyzed
surfactant was cleared by centrifugation at 15,000 rpm for 5 min
and the sample was then dried by vacuum centrifugation.
[0080] The dried sample was resuspended in 20 .mu.L 2%
acetonitrile, 0.1% formic acid, and subjected to chromatographic
separation on a Waters NanoAquity UPLC equipped with a 1.7 .mu.m
BEH130 C18 75 .mu.m I.D..times.250 mm reversed-phase column. The
mobile phase consisted of (A) 0.1% formic acid in water and (B)
0.1% formic acid in acetonitrile. Following a 1 .mu.l injection,
peptides were trapped for 5 min on a 5 .mu.m Symmetry C18 180 .mu.m
I.D..times.20 mm column at 20 .mu.L/min in 99.9% A. The analytical
column (BEH130) was then switched in-line and a linear elution
gradient of 5% B to 40% B was performed over 60 min at 400 nl/min.
The analytical column was connected to a fused silica PicoTip
emitter (New Objective, Cambridge, Mass.) with a 10 .mu.m tip
orifice and coupled to a Waters Synapt G2 HDMS QToF mass
spectrometer through an electrospray interface. The instrument was
operated in a data-dependent mode of acquisition in resolution mode
with the top three most abundant ions selected for MS/MS using a
charge state dependent CID energy setting with a 60 s dynamic
exclusion list employed.
[0081] Mass spectra were processed with Mascot Distiller (Matrix
Science) and were then submitted to Mascot searches (Matrix
Science) against a SwissProt_Ecoli database appended with the
custom Aequorea victoria GFP sequence with 10 ppm precursor and
0.04 Da product ion mass tolerances. Static mass modifications
corresponding to carbamidomethylation on Cys residues, dynamic mass
modifications corresponding to the ATRP initiator
N-(2-(2-(2-(2-aminoacetamido)acetamido)acetamido)ethyl)-2-bromo-2-methylp-
ropanamide (AEBMP), and oxidation of Met residues were included.
Searched spectra were imported into Scaffold v4.0 (Proteome
Software) and scoring thresholds were set to yield a minimum of 99%
protein confidence (implemented by the PeptideProphet algorithm)
based on decoy database searches. A minimum of two unique peptides
from each protein were required for identification. Extracted ion
chromatograms of the expected C-terminal tryptic peptide modified
by AEBMP were performed in MassLynx (v4.1) at a 20 ppm mass
accuracy window and experimental isotope distributions of the
triply charged precursor ion was compared to a theoretical isotope
distribution modeled in Molecular Weight Calculator.
Example 9: Size Exclusion Chromatography (SEC) and Conjugation
Efficiency
[0082] Analytical SEC was performed on a Shimadzu HPLC system
equipped with a UV-vis detector (SPD-10A VP) operating at 280 nm
and a fluorescence detector (RF-10Axl) set at 460 nm excitation and
507 nm emission. 30 .mu.L of samples at .about.25 .mu.M
concentration were separated on a Protein KW-803 column (with a
guard column) using Tris-HCl buffer (0.1M Tris-HCl, pH 7.4) as
mobile phase at 25.degree. C. and a flow rate of 0.5 mL/min.
Preparative SEC to purify the conjugates was performed on an AKTA
system (GE Healthcare) equipped with a photodiode detector set at
280 nm and a HiLoad 26/600 Superdex 200 PG column using PBS as
mobile phase at 4.degree. C. and a flow rate of 3 mL/min.
[0083] To determine conjugation efficiency of in situ ATRP from the
C-terminus of GFP, area under the curve (AUC) of the
GFP-C-poly(OEGMA) conjugate peak and the residual unreacted
GFP-C-Br macroinitiator peak in the chromatogram of each
polymerization reaction mixture were computed by EZStart software
(v. 7.4, Shimadzu). Sum of the areas of the two peaks corresponding
to the macroinitiator and the conjugate in each chromatogram was
regarded as 100% and % fraction of the conjugate peak was recorded
as the conjugation efficiency of that particular polymerization
reaction. Values from the three reactions were then used to
calculate the mean and standard deviation of conjugation
efficiency. The calculation was done for chromatograms detected by
both UV-vis absorbance (Table 3) and fluorescence (Table 4).
Example 10: Size Exclusion Chromatography Multi-Angle Light
Scattering (SEC-MALS)
[0084] The fluid line of the analytical HPLC system was connected
downstream in series to a DAWN HELEOS II MALS detector followed by
an Optilab T-rEX refractometer (both from Wyatt Technology). The
system was calibrated with toluene and normalized with 2.0 mg/mL
Bovine Serum Albumin (BSA). Samples were filtered with 0.1 .mu.m
filters before injection. The One-detector method involving only
the refractometer was used due to low degree of UV absorbance
detected when running poly(OEGMA) polymer. Online determination of
dn/dc was performed using built-in method "dn/dc from peak" under
the assumption of 100% mass recovery. The assumption was verified
by confirming that mass recovered as measured by online UV
detection at 280 nm and mass injected as measured by offline UV
absorbance at 280 nm using Nanodrop Spectrophotometer were in close
agreement. The full recovery of sample through the column was
likely due to presence of the stealth poly(OEGMA) polymer on the
conjugates that minimized binding to the column. The actual mass
injected was determined by lyophilization followed by weighing, and
the number was entered into ASTRA (v. 6.0, Wyatt Technology) to
compute dn/dc values of the conjugates. All results were analyzed
using ASTRA 6.0.
Example 11: Dynamic Light Scattering (DLS)
[0085] DLS was performed on a DynaPro Plate Reader (Wyatt
Technology). Samples were prepared at 25 .mu.M and filtered with
0.1 .mu.m filters before analysis. The instrument was operating at
a laser wavelength of 831.95 nm, a scattering angle of 90.degree.
C. and at 25.degree. C. Data were analyzed in Dynals mode using
Dynamics 6.12.0.3.
Example 12: Fluorescence Spectroscopy
[0086] Fluorescence spectra were recorded on a CARY Eclipse
fluorescence spectrophotometer (Varian) in scan mode at 25.degree.
C. The fluorescence of samples at a concentration of 20 .mu.M was
measured with an excitation wavelength of 460 nm and the emission
intensity was recorded from 485-530 nm.
Example 13: Production of Sortase A Substrate
[0087] As shown in FIG. 1 and set forth in Examples 14-17, a
ternary fusion protein, abbreviated as "GFP-srt-ELP", was
recombinantly expressed to serve as the sortase substrate. Here,
"srt" stands for the native SrtA recognition sequence "LPETG" (SEQ
ID NO: 2) and ELP refers to an environmentally responsive
elastin-like polypeptide (ELP; SEQ ID NO: 4) that was included in
the fusion to enable easy purification of the ternary fusion by
inverse transition cycling (ITC), a non-chromatographic protein
purification method. The recognition sequence was deliberately
located between the protein and the ELP, so that transpeptidation
by Sortase A not only attaches the initiator to GFP but also
conveniently liberates the purification tag. As transpeptidation
relies on the presence of the enzyme, cleavage did not begin until
Sortase A was added in vitro. Very little, if any, of the protein
is thus lost in vivo before purification, hence increasing the
overall product yield.
Example 14: Results of SDS-PAGE Analysis of ITC Purification of
GFP-srt-ELP
[0088] SDS-PAGE was used to analyze the ITC purification of
GFP-srt-ELP. As shown in FIG. 5(a), the only species that exhibited
inverse transition behavior and thus was purified by ITC was
GFP-srt-ELP. The lack of a free ELP band demonstrates that no
premature in vivo cleavage occurred. The fusion protein was
obtained at high purity with an excellent yield of .about.300 mg/L
from E. coli shaker flask culture.
[0089] In contrast, .about.30% of the starting GFP-intein-ELP
fusion protein was lost in the previously reported intein-mediated
initiator attachment (IMIA) method. This is because the N.fwdarw.S
acyl shift step in preparation for cleavage happens
post-translationally in vivo, and the resulting linkage is prone to
intracellular reduction in the reducing environment of the
bacterial cytosol, which in turn exposes the C-terminal thioester
and liberates the ELP. As a result, the prematurely cleaved protein
cannot be purified by ITC and an additional purification step is
required to remove the cleaved ELP. In contrast, sortase-catalyzed
initiator attachment (SCIA) occurs solely in vitro, and hence
offers greater degree of control over the reaction and product
yield.
[0090] Sortase A carrying an N-terminal hexahistidine tag
(His.sub.6-tag) was also recombinantly expressed in E. coli in high
purity and high yield (135 mg/L) by immobilized metal affinity
chromatography (IMAC). FIG. 5 (b). The ATRP initiator
N-(2-(2-(2-(2-aminoacetamido)acet-amido)acetamido)ethyl)-2-bromo-2-methyl-
propanamide (AEBMP, FIG. 1) was chemically synthesized with an
N-terminal triglycine (Gly.sub.3) motif serving as the nucleophile,
as maximum reaction rates for Sortase-Mediated Ligation (SML) have
been reported when two or more glycines are incorporated.
[0091] SCIA was then carried out at a GFP-srt-ELP:Sortase A:AEBMP
ratio of 2:1:60 (FIG. 1b). SDS-PAGE analysis of the reaction
mixture showed near complete disappearance of the GFP-srt-ELP band
close to 67 kDa, and the appearance of two bands around 39 kDa and
28 kDa, corresponding to the cleaved ELP and the macroinitiator
product, abbreviated as GFP-C-Br (FIG. 1a). A control reaction was
done using Gly.sub.3 as the nucleophile, to yield GFP-C-Gly.sub.3
as negative control for subsequent ATRP reaction. Quantification of
band intensity in SDS-PAGE showed that initiator attachment
efficiency was near quantitative (.about.95% averaged across five
samples, See Table 2 in Example 6).
Example 15: Purification and Analysis of the GFP-Initiator
(Macroinitiator) Conjugates
[0092] To purify GFP-C-Br, a His.sub.6-tag was intentionally
inserted between "srt" and ELP, such that upon transpeptidation by
Sortase A, all species except GFP-C-Br carried a His.sub.6-tag.
Consequently, elution through an IMAC column yielded pure
macroinitiator in the eluent while leaving all other unwanted
species bound to the resin. SDS-PAGE analysis (FIG. 5c) indicated
that all of the GFP-C-Br was recovered by this method. The purified
GFP-C-Br was then characterized by liquid
chromatography/electrospray-ionization mass spectrometry
(LC/ESI-MS) to confirm initiator attachment (FIG. 6a). A major peak
was detected at 28,120.4 Da, which closely agrees with the
theoretical mass of 28,123.8 Da for GFP-C-Br. To prove
site-specificity of initiator attachment, GFP-C-Br was subjected to
trypsin digestion and the peptide fragments were analyzed by
LC-MS/MS. Only the C-terminal peptide fragment was detected as a
brominated cation and its experimental isotope distribution (FIG.
2b) showed nearly perfect overlap with its theoretical distribution
(FIG. 6b). These results provided strong evidence that the
brominated ATRP initiator was solely attached to the C-terminus of
GFP by SrtA. Aside from the singly brominated C-terminal peptide,
no other derivatives were detected. In SCIA however, the lack of
any thiol group close to the initiator attachment site obviates
byproduct formation through disulfide bonding, further contributing
to higher product yield. Additionally, the absence of a thiol group
also lowers the chance of dimerization of the macroinitiator.
Example 16: ATRP Growth of Polymers from GFP-C-Br
Macroinitiators
[0093] Subsequently, in situ ATRP was performed to graft
poly(OEGMA) from GFP-C-Br (FIG. 1c). Three sets of polymerization
conditions (see Table 1 in Example 5) were investigated to
synthesize conjugates of increasing molecular weights, denoted
herein as Rxn 1, Rxn 2, and Rxn 3. Size exclusion chromatography
(SEC) was performed after ATRP to characterize the polymerization
product. SEC of the product with UV-vis absorbance detection at 280
nm (FIG. 2c) showed a single peak at an elution time of 20.6 min,
corresponding to GFP-C-Br prior to polymerization. This peak
greatly diminished after polymerization, and was accompanied by the
emergence of peaks at 17.9 min, 15.9 min, and 13.3 min,
corresponding to GFP-C-poly(OEGMA) conjugates in each of the three
reactions. The results from UV-visible spectroscopic (UV/vis)
detection were consistent with those from fluorescence detection
(FIG. 7). Integration of peak areas showed that the conjugates
constituted >90% of the polymerization product on average
(Tables 3 and 4), indicating that in situ ATRP from GFP-C-Br
proceeds with extremely high efficiency.
[0094] Table 3 shows the conjugation efficiency of in situ ATRP
from C-terminus of GFP determined by AUC of HPLC chromatograms of
three independent reactions detected by UV-vis absorbance at 280
nm. Area % was calculated by dividing area of an individual peak by
total area (sum of the two) and multiplying by 100. Averaging area
% values of GFP-C-poly(OEGMA) conjugates from three reactions gives
conjugation efficiency of 95.0.+-.2.2%.
TABLE-US-00004 TABLE 3 Conjugation efficiency of in situ ATRP
detected by UV-vis absorbance at 280 nm. Area Area % GFP- GFP- GFP-
C-poly GFP- C-poly C--Br (OEGMA) Total C--Br (OEGMA) Rxn #1 59151.0
813750.0 872901.0 6.8 93.2 Rxn #2 70996.0 1202397.0 1273393.0 5.6
94.4 Rxn #3 29056.0 1133247.0 1162303.0 2.5 97.5 Mean .+-. 95.0
.+-. 2.2 Std. Dev.
[0095] Table 4 shows the conjugation efficiency of in situ ATRP
from the C-terminus of GFP determined by AUC of HPLC chromatograms
of three attempted reactions detected by fluorescence at 460 nm
excitation and 507 nm emission. Area % was calculated by dividing
area of individual peak by total area (sum of the two peaks) and
multiplying by 100. Averaging the area % values of
GFP-C-poly(OEGMA) conjugates from three reactions yielded a
conjugation efficiency of 93.6.+-.1.9%.
TABLE-US-00005 TABLE 4 Conjugation efficiency of in situ ATRP
detected by fluorescence at 460 nm excitation and 507 nm emission.
Area Area % GFP- GFP- GFP- C-poly GFP- C-poly C--Br (OEGMA) Total
C--Br (OEGMA) Rxn #1 3235695.0 74156445.0 77392140.0 4.4 95.8 Rxn
#2 7885455.0 95738118.0 103623573.0 8.2 92.4 Rxn #3 6046562.0
77048389.0 83094951.0 7.8 92.7 Mean .+-. 93.6 .+-. 1.9 Std.
Dev.
[0096] SDS-PAGE analysis provided additional evidence for the
successful growth of poly(OEGMA) from GFP-C-Br (FIG. 5d). After
each reaction, the band corresponding to GFP-C-Br (.about.28 kDa)
decreased to a much lower intensity, accompanied by a new higher
molecular weight band corresponding to the conjugate. In contrast,
when the GFP-C-Gly3 control was used in the polymerization, or when
GFP-C-Br was physically mixed with pre-synthesized poly(OEGMA),
only a single band was observed around 28 kDa, proving that
poly(OEGMA) was only grown in situ from the C-terminal initiator
attached by SCIA.
Example 17: Characterization of Polymer-Polypeptide Conjugates
[0097] The conjugates were further characterized by light
scattering. First, size exclusion chromatography multi-angle light
scattering (SEC-MALS) was performed to determine the weight-average
molecular weight (MW) and radius of gyration (Rg) of the
conjugates. The Mw of GFP-C-Br measured 28,030 Da and the
polydispersity index (PDI) was 1.01, consistent with the
theoretical value of 28,123.8 Da and the expected monodispersity of
the macroinitiator. The molecular weights of the three conjugates
measured by SEC-MALS were 6.115.times.104 Da, 8.985.times.104 Da,
and 2.631.times.105 Da, respectively, with corresponding PDI's of
1.23, 1.26, and 1.25.
[0098] Table 5 shows the light scattering characterizations of
GFP-C-Br macroinitiator and GFP-C-poly(OEGMA) conjugates. MW, PDI
and R.sub.g were measured by SEC-MALS and R.sub.h was measured by
DLS.
TABLE-US-00006 TABLE 5 Light scattering characterizations of
GFP-C--Br macroinitiator and GFP-C-poly(OEGMA) conjugates.
GFP-C--Br Rxn 1 Rxn 2 Rxn 3 M.sub.w (kDa) 28.0 61.2 89.9 263.1 PDI
1.01 1.23 1.26 1.25 R.sub.g (nm) N/A* N/A* 10.6 19.2 R.sub.h (nm)
3.6 6.4 10.0 18.3 R.sub.g/R.sub.h N/A N/A 1.06 1.05 *Below lower
limit of detection of instrument.
[0099] These results show that by tuning the ATRP conditions,
conjugates can be synthesized from macroinitiators generated by
SCIA with different molecular weights and fairly low
polydispersity. The Rg's of GFP-C-Br and Rxn 1 conjugate could not
be accurately determined by SEC-MALS, as they fell below the 10 nm
lower limit of detection at a laser wavelength of 638 nm. R.sub.g's
of the products of Rxn 2 and 3 were 10.6 nm and 19.2 nm,
respectively. Next, the hydrodynamic radius (R.sub.h) of each
species was measured by dynamic light scattering (DLS). The Rh of
GFP-C-Br was determined to be 3.6 nm. In situ growth of poly(OEGMA)
from the macroinitiator resulted in an increase of the Rh to 6.4
nm, 10.0 nm, and 18.3 nm, for the three polymerization reactions,
respectively. With both Rg and Rh available for Rxn 2 and 3, their
corresponding R.sub.g/R.sub.h ratios (p=form factor) were
calculated, yielding values of 1.06 and 1.05, respectively. To put
these values in perspective, for globular proteins is .about.0.775,
while that of monodisperse random coil polymer in theta solvent is
1.50. An increase in polymer polydispersity and the presence in a
good solvent can increase .rho.32. Thus, their .rho. values suggest
that the overall conformation of the GFP-C-poly(OEGMA) conjugates
lies somewhere between that of their components. The conjugates
could be easily and completely purified by preparative SEC.
Fluorescence spectroscopy of unmodified GFP, GFP-C-Br, and purified
GFP-C-poly(OEGMA) clearly shows that each step in the synthesis of
the conjugate has minimal effect on the activity of the protein
(FIG. 8).
[0100] Various features and advantages of the invention are set
forth in the following claims.
Sequence CWU 1
1
12133PRTUnknownpeptide 1Asp His Met Val Leu Leu Glu Phe Val Thr Ala
Ala Gly Ile Thr His 1 5 10 15 Gly Met Asp Glu Leu Tyr Asn Val Asp
Gly Gly Gly Ser Leu Pro Glu 20 25 30 Thr 25PRTUnknownpeptide 2Leu
Pro Glu Thr Gly 1 5 35PRTUnknownpeptidemisc_feature(3)..(3)Xaa can
be any naturally occurring amino acid 3Leu Pro Xaa Thr Gly 1 5
45PRTUnknownpeptidemisc_feature(4)..(4)Xaa is alanine (A), glycine
(G), or valine (V) 4Val Pro Gly Xaa Gly 1 5 5152PRTStaphylococcus
aureus 5Gly Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys Val Ala
Gly 1 5 10 15 Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro Val
Tyr Pro Gly 20 25 30 Pro Ala Thr Pro Glu Gln Leu Asn Arg Gly Val
Ser Phe Ala Glu Glu 35 40 45 Asn Glu Ser Leu Asp Asp Gln Asn Ile
Ser Ile Ala Gly His Thr Phe 50 55 60 Ile Asp Arg Pro Asn Tyr Gln
Phe Thr Asn Leu Lys Ala Ala Lys Lys 65 70 75 80 Gly Ser Met Val Tyr
Phe Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys 85 90 95 Met Thr Ser
Ile Arg Asp Val Lys Pro Thr Asp Val Gly Val Leu Asp 100 105 110 Glu
Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp 115 120
125 Tyr Asn Glu Lys Thr Gly Val Trp Glu Lys Arg Lys Ile Phe Val Ala
130 135 140 Thr Glu Val Lys Ala Leu Val Thr 145 150
6206PRTStaphylococcus aureus 6Met Lys Lys Trp Thr Asn Arg Leu Met
Thr Ile Ala Gly Val Val Leu 1 5 10 15 Ile Leu Val Ala Ala Tyr Leu
Phe Ala Lys Pro His Ile Asp Asn Tyr 20 25 30 Leu His Asp Lys Asp
Lys Asp Glu Lys Ile Glu Gln Tyr Asp Lys Asn 35 40 45 Val Lys Glu
Gln Ala Ser Lys Asp Asn Lys Gln Gln Ala Lys Pro Gln 50 55 60 Ile
Pro Lys Asp Lys Ser Lys Val Ala Gly Tyr Ile Glu Ile Pro Asp 65 70
75 80 Ala Asp Ile Lys Glu Pro Val Tyr Pro Gly Pro Ala Thr Pro Glu
Gln 85 90 95 Leu Asn Arg Gly Val Ser Phe Ala Glu Glu Asn Glu Ser
Leu Asp Asp 100 105 110 Gln Asn Ile Ser Ile Ala Gly His Thr Phe Ile
Asp Arg Pro Asn Tyr 115 120 125 Gln Phe Thr Asn Leu Lys Ala Ala Lys
Lys Gly Ser Met Val Tyr Phe 130 135 140 Lys Val Gly Asn Glu Thr Arg
Lys Tyr Lys Met Thr Ser Ile Arg Asp 145 150 155 160 Val Lys Pro Thr
Asp Val Glu Val Leu Asp Glu Gln Lys Gly Lys Asp 165 170 175 Lys Gln
Leu Thr Leu Ile Thr Cys Asp Asp Tyr Asn Glu Lys Thr Gly 180 185 190
Val Trp Glu Lys Arg Lys Ile Phe Val Ala Thr Glu Val Lys 195 200 205
7238PRTUnknownpeptide 7Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His
Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr
Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ala Tyr Gly
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90
95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala
Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile
Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu
Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Val Leu Ser 195 200 205 Lys
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215
220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230
235 823DNAArtificial sequencesynthetic 8ttcccctcta gaaataattt tgt
23966DNAArtificial sequencesynthetic 9caagcaaagc ttaccgccgg
tttccggcaa gctgccccca ccgccgtcga cgttgtacag 60ttcatc
66105PRTUnknownsynthetic 10Gly Gly Gly Gly Ser 1 5
11456DNAStaphylococcus aureus 11ggccaagcta aacctcaaat tccgaaagat
aaatcgaaag tggcaggcta tattgaaatt 60ccagatgctg atattaaaga accagtgtat
ccaggaccag caacacctga acaattaaat 120agaggtgtaa gctttgcaga
agaaaatgaa tcactagatg atcaaaatat ttcaattgca 180ggacacactt
tcattgaccg tccgaactat caatttacaa atcttaaagc agccaaaaaa
240ggtagtatgg tgtactttaa agttggtaat gaaacacgta agtataaaat
gacaagtata 300agagatgtta agcctacaga tgtaggagtt ctagatgaac
aaaaaggtaa agataaacaa 360ttaacattaa ttacttgtga tgattacaat
gaaaagacag gcgtttggga aaaacgtaaa 420atctttgtag ctacagaagt
caaagcacta gttact 45612621DNAStaphylococcus aureus 12atgaaaaaat
ggacaaatcg attaatgaca atcgctggtg tagtacttat cctagtggca 60gcatatttgt
ttgctaaacc acatatcgat aattatcttc acgataaaga taaagatgaa
120aagattgaac aatatgataa aaatgtaaaa gaacaggcga gtaaagacaa
taagcagcaa 180gctaaacctc aaattccgaa agataaatca aaagtggcag
gctatattga aattccagat 240gctgatatta aagaaccagt atatccagga
ccagcaacac ctgaacaatt aaatagaggt 300gtaagctttg cagaagaaaa
tgaatcacta gatgatcaaa atatttcaat tgcaggacac 360actttcattg
accgtccgaa ctatcaattt acaaatctta aagcagccaa aaaaggtagt
420atggtgtact ttaaagttgg taatgaaaca cgtaagtata aaatgacaag
tataagagat 480gttaagccaa cagatgtaga agttctagat gaacaaaaag
gtaaagataa acaattaaca 540ttaattactt gtgatgatta caatgaaaag
acaggcgttt gggaaaaacg taaaatcttt 600gtagctacag aagtcaaata a 621
* * * * *