Metal-Free Polymer-Based Affinity Medium for Selective Purification of His6-Tagged Proteins

Article abstract of DOI:10.1021/acs.biomac.1c00119

We report a metal free synthetic hydrogel copolymer with affinity and selectivity for His6-tagged peptides and proteins. Small libraries of copolymers incorporating charged and hydrophobic functional groups were screened by an iterative process for His6 p

Full text of DOI:10.1021/acs.biomac.1c00119

pubs.acs.org/Biomac
Article
Metal-Free Polymer-Based Affinity Medium for Selective Purification
of His6-Tagged Proteins
Keiichi Yoshimatsu, Krista R. Fruehauf, Quanhong Zhu, Adam Weisman, Jun Fan, Min Xue,
John M. Beierle, Paul E. Rose, Jennifer Aral, Linda F. Epstein, Philip Tagari, Les P. Miranda,
and Kenneth J. Shea*
Cite This: Biomacromolecules 2021, 22, 1695−1705
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: We report a metal free synthetic hydrogel copolymer with affinity and
selectivity for His6-tagged peptides and proteins. Small libraries of copolymers
incorporating charged and hydrophobic functional groups were screened by an iterative
process for His6 peptide affinity. The monomer selection was guided by interactions
found in the crystal structure of an anti-His tag antibody-His6 peptide antigen complex.
Synthetic copolymers incorporating a phenylalanine-derived monomer were found to
exhibit strong affinity for both His6-containing peptides and proteins. The proximity of
both aromatic and negatively charged functional groups were important factors for the
His6 affinity of hydrogel copolymers. His6 affinity was not compromised by the
presence of enzyme cleavage sequences. The His6−copolymer interactions are pH
sensitive: the copolymer selectively captured His6 peptides at pH 7.8 while the
interactions were substantially weakened at pH 8.6. This provided mild conditions for
releasing His6-tagged proteins from the copolymer. Finally, a synthetic copolymer
coated chromatographic medium was prepared and applied to the purification of a His6-tagged protein from an E. coli expression
system. The results establish that a synthetic copolymer-based affinity medium can function as an effective alternative to immobilized
metal ion columns for the purification of His6-tagged proteins.
tetradentate ligand, nitrilotriacetic acid (NTA), attached to a
solid support. This system, immobilized metal ion affinity
chromatography (IMAC),26 has proven to be extremely
serviceable for affinity purification. The value of His-tags is
derived in part from their small size; the presence of the tag
exerts little disruptive effect on protein expression and
folding.27 The His-tag is broadly compatible with many
proteins and is often proceeded by an enzymatically cleavable
group for removal.28 However, IMAC is not without some
drawbacks. Histidine-rich proteins can coordinate with the
immobilized metals on the column and coelute with the
protein of interest.25,29 Problems can also arise when purifying
metalloproteins on IMAC columns due to the potential risk for
metal removal or exchange with the metal on the column.30
His-tagged proteins are frequently released from the solid
support by treatment with high concentrations of imidazole
which often necessitates an additional cleanup step.
INTRODUCTION
■
Recombinant proteins occupy an essential role in modern
biological science and biotechnology. They have revolutionized
areas of research ranging from enzyme engineering for
technical applications1 to new therapies for human diseases.2−4
To meet these expanding needs, recombinant proteins are now
produced using a number of expression systems.5,6 Regardless
of the expression system, the production and purification of
recombinant proteins are intimately linked. Indeed, the
isolation and purification protocols are as important as the
expression step itself.7,8 Fusing an affinity tag at the N- or C-
terminus of the recombinant protein is one of the techniques
that have been widely used to streamline purification,9−11
improve protein yields,12,13 prevent proteolysis,14 facilitate
protein refolding,15 reduce/increase antigenicity,16 and/or
increase solubility.17−21 However, the physical properties and
stability of the product proteins often restricts the conditions
for separation and purification. The absence of a “one-size-fits-
all” solution continues to drive the development of new
separation and purification options.9−11,22−25
Received: January 29, 2021
Revised: March 16, 2021
Published: March 30, 2021
The polyhistidine-tag appended to the N- or C-terminus of a
protein is one of the most common affinity tags in use. The
presence of multiple imidazole groups, often six histidine
residues (His6), allows for coordination to transition metal
ions (e.g., Ni2+, Co2+, Cu2+, Zn2+) immobilized with a
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705
© 2021 American Chemical Society
1695

Biomacromolecules
pubs.acs.org/Biomac
Article
(dd, J = 17.5 Hz, 10.5 Hz, 1 H, CHCH2), 6.21∼6.13 (d, J = 17.5
Hz, 1 H, CHCH2 trans), 5.62 (d, J = 10.5 Hz, 1 H, CHCH2 cis),
4.75∼4.71 (m, CHCH2), 3.25∼3.20 (m, 1 H, CH2CH), 3.01∼2.96
(m, 1 H, CH2CH). 13C NMR (125 MHz, CD3OD) δ 173.18, 166.49,
137.02, 130.18, 128.83, 128.04, 126.41, 125.85, 53.78, 37.02; HRMS
(ESI-TOF): m /z calcd for C12H13NNaO3 [M + Na]+ 242.0788;
found 242.0790.
Other studies have incorporated the basic concepts of IMAC
into synthetic polymers by utilizing NTA as a comonomer.31,32
However, these designs do not move away from the
aforementioned issues associated with IMAC. There are
commercially available anti-His tag antibodies but antibody-
immobilized columns are more costly than IMAC columns.
Along those lines, our goal was to develop a metal-free,
effective His-tag affinity material that could be used in place of
IMAC. The work builds upon our synthetic antibody program
that develops low information content statistical copolymers
engineered with high affinity and selectivity for peptides and
proteins.33,34 Here we describe an N-isopropylacrylamide
(NIPAm)-based, metal-free synthetic copolymer with affinity
and selectively for His6-tagged peptides and proteins. We
achieved this goal by incorporating both carboxylic acid and
hydrophobic groups in the hydrogel copolymer. The highest
His6-tag affinity copolymer incorporates an amino acid derived
comonomer, N-acryloyl phenylalanine (AcPhe). The perform-
ance of the hydrogel copolymer as a chromatographic medium
is demonstrated by coating a support material with the
copolymer and comparing performance with an IMAC column
for the purification of a His6-tagged protein from E. coli cell
lysates at pH 7.8.
Synthesis of N-Acryloyl L-Leucine (AcLeu). L-Leucine (2.62 g,
0.020 mol) was dissolved in 20 mL of 2 M sodium hydroxide aqueous
solution. To a well-stirred aqueous solution of L-leucine and sodium
hydroxide, acryloyl chloride (1.75 mL, 0.022 mol) was added
dropwise while the reaction mixture was kept below 0 °C by external
ice-bath cooling. After the addition, stirring was continued for
additional 2 h. The mixture was washed with ether. The mixture was
acidified to pH 2 with hydrochloric acid solution. The white semisolid
was collected by filtration. After the obtained semisolid was dissolved
with preheated water and passed through a filter membrane. The
filtrate was cooled down to yield a white product. Yield: 52.4% (1.94
1
g); H NMR (500 MHz, CD3Cl3): δ 6.85 (d, J = 8.0 Hz, 1 H, NH),
6.28 (d, J = 16.5 Hz, 1 H, CHCH2 trans), 6.19 (dd, J = 17.0 Hz, 10.5
Hz, 1 H, CHCH2), 5.67 (d, J = 10.0 Hz, 1 H, CHCH2 cis), 4.62
(m, 1 H, CH2CHNH), 1.71 (m, 2 H, CH3CH2CH), 1.64∼1.61 (m, 1
H, (CH3)2CHCH2), 0.94 (m, 6 H, (CH3)2CH); 13C NMR (125
MHz, CD3Cl3): δ 176.19, 166.02, 129.97, 127.99, 51.03, 41.22, 24.95,
22.84, 21.94. MS (ESI-TOF): m /z calcd for C9H16NO3 [M + H]+
186.2282, found 186.25; calcd for C9H15NNaO3 [M + Na]+
208.2101, found 208.25.
MATERIALS AND METHODS
Synthesis of N-Acryloyl L-Alanine (AcAla). L-Alanine (1.78 g,
0.020 mol) was dissolved in 20 mL of 2 M sodium hydroxide aqueous
solution. To a well-stirred aqueous solution of L-alanine and sodium
hydroxide, acryloyl chloride (1.75 mL, 0.022 mol) was added
dropwise while the reaction mixture was kept below 0 °C by external
ice-bath cooling. After the addition, stirring was continued for
additional 2 h at room temperature. The mixture was acidified to pH
2 with hydrochloric acid solution. A white product was gradually
■
Materials. All chemicals were obtained from commercial sources:
propargylamine, N-isopropylacrylamide (NIPAm), N-phenylacryla-
mide (PAm), ammonium persulfate, sepharose CL-4B, epichlorohy-
drin, tris(hydroxymethyl)aminomethane (Tris), and glycine were
from Sigma-Aldrich, Inc. (St. Louis, MO); β-mercaptoethanol (βME)
and dimethyl sulfoxide (DMSO) were from Sigma, Ltd.; L-alanine, L-
leucine, acrylic acid (AAc), and sodium dodecyl sulfate (SDS) were
from Aldrich Chemical Co.; N,N′-methylenebis(acrylamide) (BIS)
was from Fluka; N-tert-butylacrylamide (TBAm), sodium L-ascorbate,
and ethylenediaminetetraacetic acid (EDTA) were from Acros
Organics (Geel, Belgium); acryloyl chloride (96%, stabilized with
400 ppm phenothiazine) was from Alfa Aesar (Haverhill, MA); L-
phenylalanine was from MP Biomedicals (Santa Ana, CA); sodium
azide and sodium hydroxide (NaOH) were from Fisher Scientific;
cupper (II) sulfate (CuSO4) was from Mallinckrodt; and glycerol was
from Merck KGaA (Darmstadt, Germany).
1
formed and separated by filtration. Yield: 41.9% (1.20 g); H NMR
(500 MHz, DMSO-d6): δ 8.43 (d, J = 7.0 Hz, 1 H, NH), 6.32 (dd, J =
16.5 Hz, 10.5 Hz, 1 H, CHCH2), 6.13 (d, J = 16.5 Hz, 1 H, CH
CH2 trans), 5.64 (d, J = 10.5 Hz, 1 H, CHCH2 cis), 4.31 (t, J = 7.5
Hz, 1 H, CH3CH), 1.33 (d, J = 7.5 Hz, 1 H, CH3CH); 13C NMR
(125 MHz, DMSO-d6): δ 174.57, 164.72, 131.80, 126.14, 47.98,
17.71; MS (ESI-TOF): m /z calcd for C6H10NO3 [M + H]+
144.1485, found 144.3; calcd for C6H9NNaO3 [M + Na]+
166.1304, found 166.2.
Synthesis of N-Propargyl Acrylamide. N-Propargyl acrylamide
was synthesized by following a previously reported procedure36 with a
minor modification. Propargylamine (0.64 mL, 10 mmol) was
dissolved in 15 mL of 1 M sodium hydroxide aqueous solution. To
a well-stirred aqueous solution of propargylamine and sodium
hydroxide, acryloyl chloride (1.22 mL, 15 mmol) in 2 mL of
dichloromethane was added dropwise while the reaction mixture was
kept at 0 °C by external ice-bath cooling. After addition was complete,
stirring was continued for an additional 2 h at room temperature. The
product was extracted with 15 mL of EtOAc three times and dried
over anhydrous MgSO4. The solvent was removed on a rotary
All peptides were synthesized by solid-phase peptide synthesis.
Enhanced green fluorescence protein with hexahistidine tag (EGFP-
His6) and insulin-like growth factor 1 receptor kinase domain with
hexahistidine tag (His6-IGF1RKD) were recombinantly expressed in
E. coli. After cell lysis, the proteins were purified by using immobilized
ion affinity chromatography. The purified proteins were stored in 20
mM Tris-HCl Buffer (pH 8.0) containing 150 mM NaCl and 15%
Glycerol at 4 °C until use. Protein Assay Kit, Precast SDS-PAGE gels
(12% Mini-PROTEAN TGX) and a molecular weight marker
solution (Precision plus protein standards) were purchased from
Bio-Rad Laboratories, Inc. (Hercules, CA). NIPAm was recrystallized
from hexane before use. Other chemicals were used as-received.
Water used in polymerization and characterization was purified using
a Barnstead Nanopure Diamond system.
1
evaporator to yield a pale brown solid. Yield: 74% (814 mg); H
NMR (500 MHz, CD3OD): δ 6.30 (dd, 1 H, J = 1.1, 16.9 Hz), 6.09
(dd, 1 H, J = 10.4, 16.9 Hz), 5.93 (br, 1 H), 5.67 (dd, 1 H, J = 1.2,
10.3 Hz), 4.11 (dd, 2 H, J = 2.6, 5.3 Hz), 2.23 (t, 1 H, J = 2.4 Hz);
13C NMR (125 MHz, CD3OD): δ 165.4, 130.2, 127.6, 79.5, 72.0,
Synthesis of N-Acryloyl L-Phenylalanine (AcPhe). N-Acryloyl
L-phenylalanine was synthesized following a previously reported
procedure.35 L-Phenylalanine (3.63 g, 0.020 mol) was dissolved in 20
mL of 2 M sodium hydroxide aqueous solution. To a well-stirred
aqueous solution of L-phenylalanine and sodium hydroxide, acryloyl
chloride (1.75 mL, 0.022 mol) was added dropwise while the reaction
mixture was kept below 0 °C by external ice-bath cooling. After the
addition, stirring was continued for an additional 2 h at room
temperature. The mixture was acidified to pH 2 with hydrochloric
acid solution. A white product was separated by suction filtration and
29.5; HRMS (CI-TOF): m /z calcd for C6H8NO [M + H]+
110.0600; Found 110.0623.
Preparation of Polymer Nanoparticles (NPs) Consisting of
AAc, PAm, and/or TBAm as Comonomer(s). The procedure
reported by Debord and Lyon was adapted to synthesize polymer
nanoparticles (NPs).37−39 AAc (X mol %), PAm (Y mol %), TBAm
(Z mol %), NIPAm (98 − X − Y − Z mol %), BIS (2 mol %), and
SDS (10 mg) were dissolved in water (50 mL) and the resulting
solutions were filtered through a no. 2 Whatman filter paper. PAm or
TBAm was dissolved in acetonitrile (0.6 mL) or ethanol (1 mL),
1
purified by recrystallization from water. Yield: 45.7% (2.74 g); H
NMR (500 MHz, CD3OD) δ 7.27∼7.16 (m, 5 H, C6H5), 6.27∼6.13
1696
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
respectively, before addition to the monomer solution. The total
monomer concentration was 65 mM. Nitrogen gas was bubbled
through the reaction mixtures for 30 min. Following the addition of
ammonium persulfate aqueous solution (30 mg in 500 μL of water),
the prepolymerization mixture was sealed under nitrogen gas.
Polymerization was carried out by inserting the round bottle flask
containing prepolymerization mixture in an oil bath preset to 60 °C
for 3 h. The polymerized solutions were purified by dialysis using a
dialysis membrane (12 000−14 000 Da MWCO) against an excess
amount of pure water (changed more than twice a day) for 4 days.
The NPs with a larger diameter were prepared in the identical manner
except for omission of SDS. The prepared NPs could be stored at
room temperature without noticeable change for several weeks.
Preparation of Polymer Nanoparticles Incorporating Amino
Acid-Derived Comonomers. Amino acid-derived monomer (X mol
%), NIPAm (98 − X mol %), BIS (2 mol %), and SDS (10 mg) were
dissolved in water (50 mL) and the resulting solutions were filtered
through a no. 2 Whatman filter paper. The total monomer
concentration was 65 mM. Nitrogen gas was bubbled through the
reaction mixtures for 30 min. Following the addition of ammonium
persulfate aqueous solution (30 mg in 500 μL of water), the
prepolymerization mixture was sealed under nitrogen gas. Polymer-
ization was carried out by inserting the round-bottom flask containing
prepolymerization mixture in an oil bath preset to 60 °C for 3 h. The
polymerized solutions were purified by dialysis using a dialysis
membrane (12 000−14 000 Da MWCO) against an excess of pure
water (changed more than twice a day) for 4 days. The NPs with a
larger diameter were prepared in the identical manner except for
omission of SDS. The prepared NPs could be stored at room
temperature without noticeable change for several weeks.
containing monomers (AcPhe (40 mol %), NIPAm (58 mol %), and
BIS (2 mol %)), 2 mg of sodium dodecyl sulfate, and 6 mg of
ammonium persulfate. The total concentration of the monomers was
65 mM. The mixture was purged with dry nitrogen for 30 min and
sealed under nitrogen. The reaction was initiated by inserting the flask
into an oil bath at 60 °C and kept for 3 h with stirring. The polymer-
coated Sepharose beads were then transferred in a plastic column with
a frit and washed thoroughly with water. The prepared synthetic
copolymer-coated beads could be stored at 4 °C (in a refrigerator)
without noticeable change for several weeks.
Representative Procedure for the Quantification of Pep-
tides Bound to NPs by Centrifugal Filtration Assay. Buffer
solution (20 mM Tris-HCl (pH 7.8), 10% Tween 20 (w/v, in H2O),
peptide solution (100 μM in H2O), and NP solution (6.0 mg mL−1 in
H2O) were prepared in separate tubes. The buffer solution (180.5
μL), 10% Tween 20 (2.5 μL), peptide solution (25 μL), and NP
solution (42 μL) were mixed in centrifugal filtration tubes (Nanosep
with Omega (modified poly(ether sulfone)) ultrafiltration membrane,
MWCO: 100 kDa, PALL corp.) and incubated at room temp for 30
min. The final concentration of the buffer, peptide, and NP were ∼15
mM, 10 μM, and ∼1.0 mg mL−1, respectively. Control samples had
the NP solution replaced by an equal volume of purified water. In
order to filter off the NPs and NP-bound peptides, the solution was
passed through the membrane by the centrifugal filtration (10 000
rpm, 20 min, room temperature). Control samples had the NP
solution replaced by an equal volume of purified water and were
treated in the same manner as described above. A part of the filtrate
(100 μL) was mixed with 100 μL of 4-acetamidobenzoic acid solution
(40 μM in H2O). The amount of the peptide present in the filtrate
was quantified by HPLC (Solvents: A = H2O + 0.1% TFA, B =
MeCN + 0.1% TFA; Method: 0% B (5 min) → gradient (0% B to
50% B, over 10 min) → gradient (50% B to 100% B, over 1 min) →
100% B (5 min) → gradient (100% B to 0% B, over 1 min) → 0% B
(5 min); Injection volume: 100 μL; Flow rate: 1.2 mL/min;
Detection: absorbance at 220 nm.). The peak areas of the peptides
were integrated and normalized against the peak area of the internal
standard (4-acetamidobenzoic acid). The % of peptides bound to the
NP was calculated by the following equation:
Characterization of NPs. The hydrodynamic diameter of NPs
was determined in aqueous solution (25 0.1 °C) by dynamic light
scattering (DLS) instrument equipped with zetasizer software Ver.
6.12 (Zetasizer Nano ZS, Malvern Instruments, Ltd.). The yield and
concentration of NPs were determined by gravimetric analysis of
lyophilized aliquots of NPs.
Preparation of Polymer-Coated Sepharose CL-4B Beads.
Step i: Functionalization of Sepharose CL-4B beadsSepharose CL-4B
(40 mL in gel volume) was washed with water (120 mL) 3× and then
suspended in 25 mL of 1 M sodium hydroxide aqueous solution.
Epichlorohydrin (20 mL, 0.25 mol) was added to the mixture. The
pH of the gel suspension was ∼13. The mixture was incubated in an
oil bath at 40 °C with vigorous stirring overnight. The gel was then
thoroughly washed with Et2O, acetone, and water. The number of
epoxide groups in the epoxy-activated Sepharose was determined
using the method of Sundberg and Porath.40 Epoxy-activated
Sepharose beads (100 μL gel volume) was added to 2 mL of 1.3 M
sodium thiosulfate aqueous solution, and subsequently titrated with 1
M hydrochloric acid until pH 7.0 using phenol red as an indicator.
The amount of hydrochloric acid consumed was used to estimate the
number of epoxy groups on the Sepharose beads. The titration
indicated that 1 mL (gel volume) of the functionalized Sepharose
beads contain ∼0.04 mmol of epoxide groups. Sodium azide (4.5 g, 90
mmol) was dissolved in 100 mL of water and mixed with the epoxy-
activated Sepharose beads (40 mL in gel volume). The mixture was
incubated at room temperature with stirring overnight. The gel was
thoroughly washed with water and kept in 10 mL water until further
use. The azide-functionalized Sepharose beads (10 mL in gel volume,
containing ∼0.4 mmol of azide groups) was suspended in 14 mL of
methanol:water (1:1, v/v). To the suspension N-propargyl acrylamide
(6.6 mg, 0.6 mmol) dissolved in 2.0 mL of methanol/water (1:1, v/v),
400 μL of 50 mM sodium ascorbate aqueous solution, and 100 μL of
100 mM CuSO4 aqueous solution were added. The mixture was
incubated at room temperature under stirring overnight. The gel was
thoroughly washed once with water (200 mL), twice with 20 mM
sodium phosphate buffer (pH 7.3) containing 1 mM EDTA (200
mL), twice with 20 mM sodium phosphate buffer (pH 7.3), and twice
with water (400 mL). Step ii: Coating of Sepharose CL-4B beads with
polymer chainsThe propargyl acrylamide-functionalized Sepharose
beads (5 mL in gel volume) was suspended in 10 mL of water
NPAPS
NPAPCS
i
y
j
z
z
z
j
%PBNP = 1 −
× 100
j
k
{
where %PBNP is the percentage of peptides bound to the NP,
NPAPS is the normalized peak area of peptides in the sample, and
NPAPCS is the normalized peak area of peptides in the control
sample.
Procedure for Nonlinear Curve Fitting and Estimation of
the Average Dissociation Constant of Binding Sites on
AcPhe40 NP. The data of equilibrium binding study at different
concentrations of AcPhe40 NPs for its ability to bind Met-His6-
PreScission peptide in 15 mM Tris-HCl (pH 7.8) containing 0.1%
Tween 20 were analyzed by nonlinear curve fitting with one-site
model (eq 1).
Bmax × Bunoccupied
Kd + Bunoccupied
Boccupied
=
(1)
In order to perform curve fitting, the amounts of peptide depleted
from solution were used as Boccupied and the values calculated by the eq
2 were used as Bunoccupied
.
Bunoccupied = x − Boccupied
(2)
In each run, different values were assigned to x in eq 2, and the
values for Bmax and Kd that yield the best fit were computed by least-
squares fitting regression analysis on Nonlinear Least Squares Curve
Fitting Webtool.41 The best estimate of x and Kd values for the
binding sites with Met-His6-PreScission peptide affinity were
determined from the fitting result that returned a value that was
closest to 10 μmol binding sites/L of NP suspension, which
corresponds to the theoretical maximum in this experimental setting,
for Bmax
.
1697
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
Procedure for Quantification of EGFPs Bound to NPs by
Centrifugal Filtration Assay. Protein solution (80 μg mL−1 in 16
mM Tris-HCl (pH 7.8)), 10% Tween 20 (w/v, in H2O), and NP
solution (0.8 mg mL−1 in H2O) were prepared in separate tubes. The
protein solution (244.4 μL), 10% Tween 20 (2.5 μL), and NP
solution (3.1 μL) were mixed in centrifugal filtration tubes (Nanosep
with Omega (modified poly(ether sulfone)) ultrafiltration membrane,
MWCO: 100 kDa, PALL Corp.) and incubated at room temp for 30
min. The final concentration of the protein and NP were ∼8.0 μg
mL−1 and 10 μg mL−1, respectively. Control samples had the NP
solution replaced by an equal volume of purified water. Centrifugation
at 10 000 rpm at room temperature for 10 min filtered off the NPs
and NP-bound proteins. Control samples replaced the NP solution by
an equal volume of purified water and were treated in the same
manner as described above. The amount of EGFPs in the filtrate was
quantified by fluorescence measurements on SPEX Fluorolog
spectrofluorometer (Jobin Yvon, Edison, NJ) (Excitation wavelength:
475 nm, and Emission wavelength: the average values of 497−500
nm.). The % of EGFPs bound to the NPs was calculated by the
following equation:
sample preparation solution (7 μL; water (68 vol %), glycerol (20 vol
%), 2-mercaptoethanol (12 vol %), Tris (66.7 mM), SDS (40 mg/
mL), bromophenol blue (0.04%, w/v)) and heated at 90 °C for 5−10
min. The prepared samples (10 μL) or molecular weight marker
solution (0.7 μL) were loaded into the wells in 12% SDS-PAGE gels
immersed in a running buffer (aqueous solution containing 25 mM
Tris, 192 mM glycine and 0.1% SDS), separated by electrophoresis.
The gels were stained using the silver staining method.
RESULTS AND DISCUSSION
■
Functional Group Selection, Copolymer Synthesis,
and His6 Affinity Screen. The study begins with the
synthesis of a small library of NIPAm copolymer NPs
containing variable levels of negatively charged and hydro-
phobic comonomers. NPs of this type have been previously
reported to exhibit high water content and mesh sizes sufficient
to incorporate peptides and proteins into their interior.33 We
used directed chemical evolution, a method described
previously for the discovery of synthetic copolymer comple-
ments to biological macromolecules.34,42,43,38,44,45 The selec-
tion of monomers for inclusion in the copolymer was aided by
analysis of the crystal structure of an anti-His tag antibody-
His6 peptide antigen complex.46 As shown in Figure 1, the
i
FEIS y
j
z
z
z
j
% of EGFPs bound to the NP = 1 −
× 100
j
k
FEICS
{
where FEIS is the fluorescence emission intensity in the sample and
FEICS is the fluorescence intensity in the control sample.
Procedure for Quantification of IGF1RKD Bound to NPs by
Centrifugal Filtration Assay. Protein solution (80 μg mL−1 in 18.2
mM Tris-HCl (pH 7.8)), 10% Tween 20 (w/v, in H2O), and NP
solution (0.8 mg mL−1 in H2O) were prepared in separate tubes. The
protein solution (216.5 μL), 10% Tween 20 (2.5 μL), and NP
solution (31 μL) were mixed in centrifugal filtration tubes (Nanosep
with Omega (modified poly(ether sulfone)) ultrafiltration membrane,
MWCO: 100 kDa, PALL corp.) and incubated at room temp for 30
min. The final concentration of the protein and NP were ∼8.0 μg
mL−1 and 100 μg mL−1, respectively. Control samples had the NP
solution replaced by an equal volume of purified water. Centrifugation
at 10 000 rpm at room temperature for 5 min resulted in the NPs and
NP-bound proteins being filtered off. Control samples replaced the
NP solution by an equal volume of purified water and were treated in
the same manner as described above. The amount of IGF1RKD in the
filtrate was analyzed by SDS-PAGE.
Chromatographic Purification of EGFP-His6 Using Polymer-
Coated Agarose Beads. A Polyprep column (Bio-Rad) was packed
with 500 μL (in gel vol.) of the polymer-coated Sepharose CL-4B
beads. The column was pre-equilibrated with by applying 10 mL of
loading buffer (20 mM Tris-HCl (pH 7.8)). The EGFP-His6 solution
(0.225 mL), an E. coli cell lysate solution (1 mL) and loading buffer
(18.3 mL) were mixed and loaded to the column. Subsequently, 10
mL of wash buffer (20 mM Tris-HCl (pH 7.8)) was applied to the
column to remove proteins weakly bound to the column. Finally,
EGFP-His6 was eluted with 20 mM Tris-HCl (pH 8.6) (4 mL × 1
time) followed by 20 mM Tris-HCl (pH 9.0) (4 mL × 4 times).
In order to compare the effectiveness of polymer-coated agarose
beads with a commercially available immobilized metal ion affinity
column, a mixture of EGFP-His6 (0.225 mL), E. coli lysate (1 mL),
and 25 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl and 10
mM imidazole (18.3 mL) was prepared and loaded to a Polyprep
column packed with 500 μL (in gel vol.) of His-Pur Ni2+-NTA
agarose beads (Thermo Scientific). In this experiment, 25 mM Tris-
HCl buffer (pH 8.0) containing 0.5 M NaCl and 20 mM imidazole
(10 mL) was applied to the column in the wash step and 25 mM Tris-
HCl buffer (pH 8.0) containing 0.3 M NaCl and 150 mM imidazole
(4 mL × 5 times) was used to elute EGFP-His6 from the column.
All fractions from loading, wash, and elution steps were analyzed by
SDS-PAGE and fluorescence measurements on Gemini XPS
fluorescence microplate reader (Molecular Devices, LLC, Sunnyvale,
CA) (Excitation wavelength at 475 nm, emission wavelength at 505
nm with a cutoff filter for 495 nm).
Figure 1. Binding pocket of anti-His tag antibody and C-terminus of
His6 peptide antigen in the crystal structure of the antibody (PDB
entry: 1KTR).46 The negative and positive Coulombic potentials on
the antibody surface are colored with red and blue, respectively. The
peptide is colored by atom type C (gray), N (blue), and O(red).
binding pocket of the antibody consists predominantly of
negatively charged areas and a nonpolar domain. Hydrophobic
interactions are observed mainly between aromatic side chains
of the antibody (Tyr, Phe) and the imidazole rings of the
antigen. Additional hydrophobic interactions are observed
between the aromatic ring of Phe and the main-chain atoms of
His6 antigen. Acidic side chains in the binding cleft of the
antibody provide a strong negative surface potential. The
charged groups act as hydrogen bond acceptors for the
nitrogen-protons of the antigen imidazole side chains. With
this information, we synthesized a library incorporating varying
SDS-PAGE Analysis. The filtrates from centrifugal filtration assays
or fractions from chromatographic study (7 μL) were mixed with a
1698
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
a
Table 1. Polymer NPs Used in the First Round of Screening
b
polymer NP
AAc(mol %)
PAm(mol %)
TBAm(mol %)
NIPAm(mol %)
BIS(mol %)
DH (nm)
AAc5/PAm20
AAc5/PAm40
AAc20/PAm20
AAc20/PAm40
AAc5/TBAm40
AAc20/TBAm40
PAm20
PAm40
TBAm40
NIPAm
5
5
20
20
5
20
40
20
40
73
53
58
38
53
38
78
58
58
98
2
2
2
2
2
2
2
2
2
2
127
108
100
74
85
74
109
85
75
40
40
20
20
40
40
431
a
b
The mol % of each monomer is in the feed ratio. Hydrodynamic diameter of NPs determined by dynamic light scattering measurement.
amounts of acrylic acid (AAc) and N-tert-butylacrylamide
(TBAm) or N-phenylacrylamide (PAm) monomers into
NIPAm-based NPs. Ammonium persulfate was used as an
initiator and all polymerization reactions were carried out in
unbuffered aqueous solution at 60 °C. In order to control the
size of NPs, a small amount of sodium dodecyl sulfate (SDS,
0.2 mg/mL) was also added to the polymerization mixture.
AAc was selected as the carboxylic acid-containing comonomer
for its hydrogen bond forming properties and negative charge
near physiological pH (pH 7.8).47,48 The aliphatic and
aromatic hydrophobic comonomers, TBAm and PAm, were
used to evaluate their relative effectiveness for binding to the
His-rich peptide. The library was then screened for binding to
a Met-His6-PreScission peptide (MHHHHHHLEVLFQGP-
CONH2). This peptide contains a common combination of N-
terminus His6-tag and cleavage site of PreScission protease
(LEVLFQGP).28 Since many proteins have narrow stability
windows, we desired to develop an affinity material that
functioned as close to the physiological pH as possible. The
monomer compositions of the NPs that were used in this work
are summarized in Table 1.
His6-tags, a finding consistent with the antibody-His6 antigen
complex structure analysis described above.46
In contrast, polymer NPs that contain only hydrophobic
monomers (TBAm or PAm) did not adsorb Met-His6-
PreScission peptide. Among the screened NPs, AAc20/
PAm20 and AAc20/PAm40 NPs showed the highest binding
capacity for Met-His6-PreScission. For example, AAc20/
PAm40 NPs (250 μg in 250 μL solution) bound 2.83 μg out
of the 4.63 μg of peptides originally added to the solution,
∼60% of the total. The result suggests aromatic hydrophobic
groups and anionic carboxylic acid groups participate
cooperatively in Met-His6-PreScission peptide binding at
pH 7.8. We also observe that the ratio of these two functional
groups is important. For example, changing from AAc5/
PAm20 to AAc20/PAm20 and from AAc5/PAm40 to AAc20/
PAm40 produce significant increases in peptide binding. In
both cases, the change in ratio has a significant effect on
peptide binding. Although these trends suggest further
optimization, we point out that we are unable to explore all
compositional space because of concern with the stability of
NPs in aqueous solution. Although the data may suggest
further increasing PAm content, our previous study showed
that at high hydrophobic monomer content one encounters
NP instability and/or clustering of hydrophobic groups within
the NP, compromising their effectiveness for interacting with
peptides or proteins.40
The binding of Met-His6-PreScission peptide to the library
of NPs in 15 mM Tris-HCl buffer containing 0.1% Tween 20
(w/v) was evaluated by incubation of NPs (1000 μg/mL) and
peptides (10 μM) in buffer at room temperature followed by
centrifugal filtration (Table 2). Unbound peptides in the
Contribution of Amino Acid-Derived Bifunctional
Comonomers to His6 Binding. Negatively charged and
hydrophobic groups were found to be important for binding to
Met-His6-PreScission peptide. Within the synthetic copoly-
mer, these two functional groups are statistically distributed
throughout the polymer network. These same functional
groups are also responsible for binding in the antibody-His6
peptide complex, but in the protein−peptide complex the two
groups can be found in close proximity. Indeed, the presence
of charged and hydrophobic groups in close proximity is a
structural motif found in many “hot spots” sites where alanine
mutations cause a significant increase in binding free energy
(greater than 2 kcal/mol).49,50 To evaluate if proximity of
hydrophobic and charged groups could result in enhanced
binding, we examined the effect of amino acid-derived
bifunctional comonomers containing both groups within a
single monomer unit.51,35,52 To this point, we synthesized and
evaluated a small library of polymer NPs that incorporated
three amino acid-derived comonomers, N-acryloyl phenyl-
alanine (AcPhe), N-acryloyl leucine (AcLeu), and N-acryloyl
alanine (AcAla). The size and nature of the hydrophobic group
was of particular interest, thus a phenyl group, isobutyl group,
Table 2. Peptide Sequences Used in This Study
peptide
sequence
Met-His6-PreScission
His6-PreScission
His4-PreScission
His2-PreScission
Met-His6-TEV
MHHHHHHLEVLFQGP-CONH2
HHHHHHLEVLFQGP-CONH2
HHHHLEVLFQGP-CONH2
HHLEVLFQGP-CONH2
MHHHHHHENLYFQG-CONH2
MHHHHHHDEVD-CONH2
Met-His6-Caspase3
filtrates were quantified using HPLC (Figure 2). The addition
of 0.1% Tween 20 was necessary to mitigate the adsorption of
peptides on the size exclusion membrane. The results establish
that AAc incorporation is required for measurable levels of
peptide binding. It was also found that NPs containing both
PAm and AAc (AAc5/PAm20, AAc20/PAm20, and AAc20/
PAm40 NPs) are more effective at binding to His6-PreScission
peptide compared to the NPs containing comparable amounts
of TBAm and AAc. This suggests that an aromatic hydro-
phobic functional group may be advantageous for affinity of
1699
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
Figure 2. Binding of Met-His6-PreScission peptide (MHHHHHHLEVLFQGP-CONH2) to polymer NPs incorporating various comonomers in
15 mM Tris-HCl buffer (pH 7.8) containing 0.1% Tween 20 (w/v). Concentration of peptide = 10 μM (18.55 μg/mL). Concentration of polymer
nanoparticles = 1000 μg/mL.
a
Table 3. Polymer NPs Used in the Second Round of Screening
b
polymer NP
AcPhe(mol %)
AcLeu(mol %)
AcAla(mol %)
NIPAm(mol %)
BIS(mol %)
DH (nm)
AcPhe20
AcPhe40
AcLeu20
AcLeu40
AcAla20
AcAla40
20
40
78
58
78
58
78
58
2
2
2
2
2
2
106
94
81
76
817
1232
20
40
20
40
a
b
The mol % of each monomer is in the feed ratio. Hydrodynamic diameter of NPs determined by dynamic light scattering measurement.
and methyl group were compared in this series. The
composition of these copolymers can be found in Table 3.
As shown in Figure 3, the polymer NP incorporating 40 mol
% of AcPhe (AcPhe40 NP) was identified as the most effective
binding curve (Figure S1 of the Supporting Information, SI).
The curve fitting analysis gave 14.7 nmol binding sites/mg
polymer and ∼300 nM as the estimated values for the number
of the binding sites and average Kd reported anti-His tag
antibody 3D5 scFv binds the His-tagged peptide with a Kd of
nmol His tag peptide/mg of scFv. Therefore, despite the fact
that these synthetic materials present a range of affinities, the
ensemble of binding sites on AcPhe40 NP exhibits a
performance that is comparable to its antibody counterpart
in terms of the numbers of binding sites (per weight basis) and
average affinity of the binding sites.
To investigate the correlation between the number of
consecutive histidine residues and the binding affinity as a
function of the AcPhe comonomer percentage, the binding of
three linear peptides, His6-PreScission, His4-PreScission, and
His2-PreScission containing six, four, or two histidine residues
with a protease cleavage site, were tested against AcPhe40 and
AcPhe20 NPs. A polymer NP that does not contain the AcPhe
comonomer was also included. As seen in Figure 4, binding
tracks with increasing histidine residues especially as the
content of AcPhe in NPs increased. This result clearly
demonstrates that the histidine residues are the major
contributors to NP binding. Additionally, interactions53,54
between multiple imidazole rings and the NP functional groups
are important for strong NP-peptide binding. It is noteworthy
that anti-His tag antibody 3D5 binds with His6, His4, and His3
peptides with equal strength.46 In contrast, AcPhe40 NP binds
more strongly with His6 peptide compared to His4, suggesting
that the AcPhe40 NP interacts with a longer stretch of
histidine residues than the biological antibody counterpart.
In some cases, the His6-tags are removed before the target
can be characterized or applied. Conventional methods include
tag removal by highly specific proteases.28 The preceding
results showed that the AcPhe40 and AcPhe20 NPs exhibit
Figure 3. Binding of Met-His6-PreScission peptide (MHHH-
HHHLEVLFQGP-CONH2) to polymer NPs incorporating 20 mol
% or 40 mol % of AcPhe, AcLeu, or AcAla comonomers in 15 mM
Tris-HCl buffer (pH 7.8) containing 0.1% Tween 20 (w/v).
Concentration of peptide = 10 μM (18.55 μg/mL). Concentration
of polymer NPs = 1000 μg/mL.
candidate. This NP absorbs >90% of the peptide added. NPs
incorporating 20 mol % of AcPhe comonomer (AcPhe20 NP),
40 mol % of AcLeu comonomer (AcLeu40 NP) or AcAla
comonomer (AcAla40 NP) absorbed less than 50% of the
peptides added to the solution. It is clear the importance of
proximity of charged and aromatic functional groups.
In an effort to compare AcPhe40 NP and a previously
reported anti-His tag antibody single-chain variable fragment
(scFv), we performed a nonlinear curve fitting analysis of the
1700
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
F i g u r e 5 . B i n d i n g o f M e t - H i s 6 - P r e S c i s s i o n
(MHHHHHHLEVLFQGP-CONH2) to AcPhe40 and AcPhe20
NPs in 15 mM Tris-HCl buffer (pH 7.0, 7.8, or 8.6) containing
0.1% Tween 20 (w/v). Concentration of peptide = 10 μM (18.55 μg/
mL). Concentration of polymer NPs = 1000 μg/mL.
Figure 4. Binding of peptides containing different numbers of
histidine residues to AcPhe40, AcPhe20, and NIPAm NPs that
incorporate 40 mol %, 20 mol %, and 0 mol % of AcPhe comonomer,
respectively, in 15 mM Tris-HCl buffer (pH 7.8) containing 0.1%
Tween 20 (w/v). Concentration of peptide = 10 μM (18.55 μg/mL).
Concentration of polymer nanoparticles = 1000 μg/mL.
The pH sensitivity may be attributed to the average number
of positive charges on His6-tag. The pKa of the histidine side
chain is ∼6. The number of formal charges present in His6-
tags can be roughly estimated to +0.6, + 0.095, and +0.015,
respectively, at pH 7.0, 7.8, and 8.6. Thus, the His-tags lose
their partial positive charge over a narrow range of pH.
Although hydrophobic domains such as phenyl rings on AcPhe
monomer units could interact with the imidazole ring via π−π
interactions, without electrostatic interactions with the anionic
carboxylic acid, the affinity is weakened. This explanation
appears to be consistent with the pH responsivity of the anti-
His tag antibody 3D5 single-chain fragment.55
Binding of His6-Tagged Proteins to AcPhe40 NPs.
The goal of this work was to develop an effective metal-free
affinity medium for purification of His6-tagged proteins from
protein expression systems. AcPhe40 NP was the most
promising candidate. We chose enhanced green fluorescence
protein (EGFP) and insulin-like growth factor 1 receptor
kinase domain (IGF1RKD) for this study. Both expressed
proteins were fused with His6-tags. A centrifugal filtration
assay procedure was used to evaluate the polymer NP−peptide
interactions for this study. As shown in Figures 6 and 7 both
strong affinity for the peptide containing the PreScission
protease cleavage site. Since the protease cleavage sequence
could affect the copolymer affinity to the His6 sequence, we
compared binding of AcPhe40 NP to two additional common
protease cleavage sequences fused with affinity tags, Met-His6-
TEV (ENLYFQG) and Met-His6-Caspase3 (DEVD). As
shown in Table 4, AcPhe40 NP was effective for binding all
three His6 containing peptides although Met-His6-Caspase3
showed a slightly lower level of binding. This could be due to
local electrostatic repulsions between the negative charges on
AcPhe40 NP and the negative charges on aspartate and
glutamate residues on the Caspase-3 cleavage site (DEVD) at
pH 7.8. The cleavage sequences for PreScission protease
(LEVLFQGP) and TEV protease (ENLYFQG) contain only 1
negatively charged residue and could account for the stronger
NP binding of Met-His6-PreScission and Met-His6-TEV in
comparison to Met-His6-Caspase3.
After establishing that the primary basis of AcPhe40 NPs
affinity was the His6-tag, the conditions required for peptide
release from the NPs were explored. In IMAC, elution of His-
tagged proteins is often achieved by competitively displacing
the His-tagged protein from immobilized metal-ions by high
concentration (250 mM or higher) of imidazole. These
conditions require a subsequent purification step. To circum-
vent this, we investigated if NP-His6 interactions can be
altered by small changes in pH. It was found that an increase in
pH is an effective means of weakening the copolymer−peptide
interactions (Figure 5). At neutral pH (7.0), both AcPhe40
and AcPhe20 NPs captured most of Met-His6-PreScission
peptide in the solution. However, as the pH is increased
slightly to 7.8, binding of AcPhe20 NP to the peptide falls off
significantly. The affinity of AcPhe40 NP for the His6 peptide
drops off at pH 8.6. We can use this pH responsiveness to
trigger the release of captured proteins from the synthetic
copolymer NPs.
Figure 6. Result of centrifugal filtration assay for evaluating the
binding of EGFP-His6 to AcPhe40 NP in 15 mM Tris-HCl buffer
(pH 7.8) containing 0.1% Tween 20 (w/v) and subsequent
centrifugal filtration. Concentration of protein (EGFP-His6 or
EGFP) = 8 μg/mL (0.24 μM). Concentration of AcPhe40 NP =
10 μg/mL.
Table 4. Binding of His6 Containing Peptides Fused with Different Protease Cleavage Sequences to AcPhe40 NP in 15 mM
a
Tris-HCl Buffer (pH 7.8) Containing 0.1% Tween 20 (w/v)
peptide
sequence
% peptide bound to AcPhe40 NP
94.5 0.8%
Met-His6-PreScission
Met-His6-TEV
MHHHHHHLEVLFQGP-CONH2
MHHHHHHENLYFQG-CONH2
MHHHHHHDEVD-CONH2
96.4 3.2%
Met-His6-Caspase
72.1 7.2%
a
Concentration of peptide = 10 μM. Concentration of polymer nanoparticles = 1000 μg/mL.
1701
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
Table 5. Amount of EGFP-His6 Found in Each Fraction
a
from the Chromatographic Study
amount of EGFP found
(% vs loaded protein mixture sample)
fraction
HisPur Ni-NTA
AcPhe40-coated Sepharose CL-4B
flow-through
wash
3.1
0.3
2.4
1.7
elution 1
elution 2
elution 3
elution 4
elution 5
23.7
21.7
14.6
7.9
38.1
20.5
13.8
3.2
5.3
2.4
a
The amount of EGFP was determined based on the fluorescence
Figure 7. SDS-PAGE analysis of filtrates from the centrifugal filtration
assay for evaluating the binding of His6-IGF1RKD to AcPhe40 NP in
15 mM Tris-HCl buffer (pH 7.8) containing 0.1% Tween 20 (w/v).
Concentration of protein (His6-IGF1RKD or IGFRKD) = 8 μg/mL
(0.24 μM). Concentration of AcPhe40 NP = 100 μg/mL. Lanes M:
molecular weight markers. Lane 1: His6-IGF1RKD-His6 control
(without filtration). Lane 2: His6-IGF1RKD + AcPhe40 NP
(filtrate). Lane 3: His6-IGF1RKD without NP (filtrate). Lane 4:
IGF1RKD control (without filtration). Lane 5: IGF1RKD + AcPhe40
NP (filtrate). Lane 6: IGF1RKD without NP (filtrate). The gel image
was taken after silver staining.
intensity (Ex. 475 nm, Em. 505 nm, cutoff filter 495 nm) and volume
of each fraction.
Tris-HCl buffer with slightly higher pH (pH 8.6 and 9.0). The
amount of EGFP in each fraction was then determined by
fluorescence measurements. The result showed the AcPhe40-
coated Sepharose CL-4B column strongly retained the EGFP-
His6 throughout the sample loading and wash steps. The
elution condition used in this experiment allowed for recovery
of up to 78% of EGFP-His6 from the AcPhe40-coated
Sepharose CL-4B column. Figure 8 shows the SDS-PAGE
EGFP-His6 and His6-IGF1RKD were taken up by AcPhe40
NP. In contrast, neither EGFP nor IGF1RKD proteins lacking
the His6-tag bound to AcPhe40 NP. Control experiments
were carried out in the absence of polymer NPs confirmed that
the depletion of proteins from solution was due to the
absorption by the polymer NPs and not caused by adhesion to
the filter membrane.
Chromatographic Purification of EGFP-His6. The use
of the centrifugal filtration procedure allowed for demonstrat-
ing small scale (250 μL) capture of His6-tagged proteins.
However, this procedure has limitations in terms of scalability.
When the experiment is performed at higher NP concen-
trations or with larger sample volumes, the NPs clogged the
pores on the filtration membrane after only a portion of the
sample solution passed through the filtration unit. To
overcome this problem, we prepared agarose beads coated
with a layer of polymer chains consisting of the same monomer
composition as AcPhe40 NP to evaluate the utility of the
copolymer-coated agarose gels for isolating EGFP-His6 from
other proteins that are derived from E. coli cells. The polymer-
coated agarose beads were prepared by the introduction of
covalently linked double bonds to the agarose beads via the
introduction of epoxide group, azidation,40 and copper-
catalyzed “click” reaction,56 followed by copolymerization of
comonomers in the presence of modified beads. This
procedure was developed to attach polymer chains to agarose
beads without introducing any undesired charged groups. The
result of elemental analysis indicated that at least 10% (in
weight %) of the polymer-coated beads consists of copolymer
chains (Table S2). His-Pur Ni2+-NTA resin was used for a
direct comparison with a commercially available affinity
medium for Ni2+-NTA-based IMAC.
Figure 8. SDS-PAGE analysis of fractions from a chromatographic
study. Lanes M: molecular weight markers. Lanes S: protein mixture
sample prior to chromatographic separation. Lane 1: flow-through
fraction from HisPur Ni-NTA column. Lane 2: wash fraction from
HisPur Ni-NTA column. Lane 3: 1st to 3rd elution fractions from
HisPur Ni-NTA column Three fractions were combined prior to the
analysis. Lane 4: flow-through fraction from AcPhe40-coated
Sepharose CL-4B column. Lane 5: wash fraction from AcPhe40-
coated Sepharose CL-4B column. Lane 6: 1st to 3rd elution fractions
from AcPhe40-coated Sepharose CL-4B column. Three fractions were
combined prior to the analysis. The gel image was taken after silver
staining.
analysis of flow-through, wash, and combined elution fractions
1−3. Most proteins derived from E. coli host cells were found
in the flow-through fraction, and the elution fractions
contained highly purified EGFP-His6. The result establishes
that AcPhe40-coated Sepharose CL-4B beads are effective for
separating EGFP-His6 from complex matrixes (E. coli cell
lysates). The purity of recovered EGFP-His6 was comparable
to or slightly higher than the sample purified by Ni2+-NTA
Table 5 summarizes the result of the chromatographic study.
In this study, a protein mixture sample containing E. coli cell
lysate and EGFP-His6 were diluted in 20 mM Tris-HCl buffer
(pH 7.8) and loaded on the column. After proteins loosely
bound to the column were washed off with 20 mM Tris-HCl
buffer (pH 8.0), EGFP-His6 was eluted by applying 20 mM
1702
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
agarose beads. Overall, fluorescence measurements and SDS-
PAGE analysis showed that AcPhe40-coated Sepharose CL-4B
column functions as effectively as a Ni-NTA column (HisPur)
demonstrating the practical utility of the AcPhe40 copolymer.
Krista R. Fruehauf − Department of Chemistry, University of
California, Irvine, California 92697, United States
Quanhong Zhu − Department of Chemistry, University of
California, Irvine, California 92697, United States
Adam Weisman − Department of Chemistry, University of
California, Irvine, California 92697, United States
Jun Fan − Department of Chemistry, University of California,
Irvine, California 92697, United States; orcid.org/0000-
0003-2986-8551
Min Xue − Department of Chemistry, University of California,
Irvine, California 92697, United States; orcid.org/0000-
0001-9786-8225
John M. Beierle − Department of Chemistry, University of
California, Irvine, California 92697, United States
Paul E. Rose − Therapeutic Discovery, Amgen Inc., Thousand
Oaks, California 91320, United States
CONCLUSIONS
■
A metal-free, selective affinity material for the His6-tag has
been developed. The material, a hydrogel copolymer, was
identified from a screen of small libraries of hydrogel
copolymer NPs. The choice of comonomers for the polymer
libraries was guided by analysis of the X-ray crystal structure of
the anti-His tag antibody 3D5 single-chain fragment
complexed to its antigen. The structural biology study
indicated both negatively charged and hydrophobic groups
would be important for affinity to the His6 antigen. Initial
screening of synthetic polymer-based NPs showed that NPs
incorporating both acrylic acid (AAc) and N-phenylacrylamide
(PAm) exhibit moderate His6-tag affinity. Further refinement
incorporating bifunctional amino acid-derived monomers
revealed that the proximity of both aromatic group and
negative charge in a phenylalanine-derived monomer was
found to be most effective. A lightly cross-linked NIPAm based
hydrogel copolymer incorporating 40% of N-acryloyl phenyl
alanine (AcPhe40), exhibited high affinity for both His6-
tagged peptides and proteins in 15 mM Tris-HCl buffer at pH
7.8. Affinity was relatively insensitive to the inclusion of
enzyme cleavage sequence. It was also found that the His6-tag
affinity of AcPhe40 NP is pH sensitive and is substantially
weakened at pH 8.6. The pH responsiveness was used to
release His6-tagged proteins from the copolymer. Column
chromatography experiments with AcPhe40-coated agarose
beads efficiently captured and released a His6-tagged EGFP
(EGFP-His6) from E. coli cell lysate establishing the potential
synthetic polymer-based affinity materials for purification of
His6-tagged proteins. The affinity material does not rely on
metal-histidine coordination, and therefore offers potential
advantages over IMAC.
Jennifer Aral − Therapeutic Discovery, Amgen Inc., Thousand
Oaks, California 91320, United States
Linda F. Epstein − Therapeutic Discovery, Amgen Inc.,
Thousand Oaks, California 91320, United States
Philip Tagari − Therapeutic Discovery, Amgen Inc., Thousand
Oaks, California 91320, United States
Les P. Miranda − Therapeutic Discovery, Amgen Inc.,
Thousand Oaks, California 91320, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.1c00119
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Prof. Charles G. Glabe (UCI) for providing access to
a fluorescence microplate reader.
■
ABBREVIATIONS
■
AAc
acrylic acid
N-acryloyl L-alanine
N-acryloyl L-leucine
N-acryloyl L-phenylalanine
N,N′-methylenebisacrylamide
enhanced green fluorescence protein
AcAla
AcLeu
AcPhe
BIS
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.1c00119.
■
sı
EGFP
IGF1RKD insulin-like growth factor 1 receptor kinase domain
Procedure for NMR measurements, summary of NP
characterization data, schematic illustration of steps in
the preparation of polymer-coated Sepharose CL-4B
beads, elemental analysis data, equilibrium binding curve
of AcPhe40 NP for Met-His6-PreScission peptide, and
1H and 13C NMR spectra (PDF)
IMAC
NIPAm
NP
NTA
PAm
SDS
TBAm
immobilized metal ion affinity chromatography
N-isopropyl acrylamide
nanoparticle
nitrilotriacetic acid
N-phenyl acrylamide
sodium dodecyl sulfate
N-tert-butyl acrylamide
AUTHOR INFORMATION
Corresponding Author
Kenneth J. Shea − Department of Chemistry, University of
California, Irvine, California 92697, United States;
orcid.org/0000-0001-5926-0059; Email: kjshea@
uci.edu
■
REFERENCES
■
(1) Arnold, F. H. Directed Evolution: Bringing New Chemistry to
Life. Angew. Chem., Int. Ed. 2018, 57, 4143−4148.
(2) Leader, B.; Baca, Q. J.; Golan, D. E. Protein Therapeutics: A
Summary and Pharmacological Classification. Nat. Rev. Drug Discovery
2008, 7, 21−39.
(3) Dimitrov, D. S. Therapeutic Proteins. In Therapeutic Proteins:
Methods and Protocols; Voynov, V., Caravella, J. A., Eds.; Methods Mol.
Biol.; Humana Press: Totowa, NJ, 2012; pp 1−26.
(4) Lagassé, H. A. D.; Alexaki, A.; Simhadri, V. L.; Katagiri, N. H.;
Jankowski, W.; Sauna, Z. E.; Kimchi-Sarfaty, C. Recent Advances in
(Therapeutic Protein) Drug Development. F1000Research 2017, 6,
113.
Authors
Keiichi Yoshimatsu − Department of Chemistry, University of
California, Irvine, California 92697, United States;
Department of Chemistry, Missouri State University,
Springfield, Missouri 65897, United States; orcid.org/
0000-0002-1428-0029
1703
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
(5) Brondyk, W. H. Chapter 11. Selecting an Appropriate Method
for Expressing a Recombinant Protein. In Methods in Enzymology;
Burgess, R. R., Deutscher, M. P., Eds.; Guide to Protein Purification,
2nd ed.; Academic Press: New York, 2009; Vol. 463, pp 131−147.
(6) Oliveira, C.; Domingues, L. Guidelines to Reach High-Quality
Purified Recombinant Proteins. Appl. Microbiol. Biotechnol. 2018, 102,
81−92.
(24) Pina, A. S.; Lowe, C. R.; Roque, A. C. A. Challenges and
Opportunities in the Purification of Recombinant Tagged Proteins.
Biotechnol. Adv. 2014, 32, 366−381.
(25) Rowinska-Zyrek, M.; Witkowska, D.; Potocki, S.; Remelli, M.;
Kozlowski, H. His-Rich Sequences − Is Plagiarism from Nature a
Good Idea? New J. Chem. 2013, 37, 58−70.
(26) Bornhorst, J. A.; Falke, J. J. Purification of Proteins Using
Polyhistidine Affinity Tags. In Methods Enzymol; Applications of
Chimeric Genes and Hybrid Proteins Part A: Gene Expression and
Protein Purification; Academic Press: New York, 2000; Vol. 326, pp
245−254.
(27) Carson, M.; Johnson, D. H.; McDonald, H.; Brouillette, C.;
DeLucas, L. J. His-Tag Impact on Structure. Acta Crystallogr., Sect. D:
Biol. Crystallogr. 2007, 63, 295−301.
(28) Waugh, D. S. An Overview of Enzymatic Reagents for the
Removal of Affinity Tags. Protein Expression Purif. 2011, 80, 283−293.
(29) Westra, D. F.; Welling, G. W.; Koedijk, D. G. A. M.; Scheffer, A.
J.; The, T. H.; Welling-Wester, S. Immobilised Metal-Ion Affinity
Chromatography Purification of Histidine-Tagged Recombinant
Proteins: A Wash Step with a Low Concentration of EDTA. J.
Chromatogr., Biomed. Appl. 2001, 760, 129−136.
(30) Maret, W. Metalloproteomics, Metalloproteomes, and the
Annotation of Metalloproteins. Metallomics 2010, 2, 117−125.
(31) Liu, J.; Spulber, M.; Wu, D.; Talom, R. M.; Palivan, C. G.;
Meier, W. Poly(N-Isopropylacrylamide-Co-Tris-Nitrilotriacetic Acid
Acrylamide) for a Combined Study of Molecular Recognition and
Spatial Constraints in Protein Binding and Interactions. J. Am. Chem.
Soc. 2014, 136, 12607−12614.
(32) Mattiasson, B.; Kumar, A.; Ivanov, A. E.; Galaev, I. Y. Metal-
Chelate Affinity Precipitation of Proteins Using Responsive Polymers.
Nat. Protoc. 2007, 2, 213−220.
(33) Luchini, A.; Geho, D. H.; Bishop, B.; Tran, D.; Xia, C.; Dufour,
R. L.; Jones, C. D.; Espina, V.; Patanarut, A.; Zhou, W.; Ross, M. M.;
Tessitore, A.; Petricoin, E. F.; Liotta, L. A. Smart Hydrogel Particles:
Biomarker Harvesting: One-Step Affinity Purification, Size Exclusion,
and Protection against Degradation. Nano Lett. 2008, 8, 350−361.
(34) O’Brien, J.; Shea, K. J. Tuning the Protein Corona of Hydrogel
Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity
Reagents. Acc. Chem. Res. 2016, 49, 1200−1210.
(35) Casolaro, M.; Paccagnini, E.; Mendichi, R.; Ito, Y. Stimuli-
Responsive Polymers Based on L-Phenylalanine Residues: Protonation
Thermodynamics of Free Polymers and Cross-Linked Hydrogels.
Macromolecules 2005, 38, 2460−2468.
(36) Malkoch, M.; Thibault, R. J.; Drockenmuller, E.;
Messerschmidt, M.; Voit, B.; Russell, T. P.; Hawker, C. J. Orthogonal
Approaches to the Simultaneous and Cascade Functionalization of
Macromolecules Using Click Chemistry. J. Am. Chem. Soc. 2005, 127,
14942−14949.
(37) Debord, J. D.; Lyon, L. A. Synthesis and Characterization of
pH-Responsive Copolymer Microgels with Tunable Volume Phase
Transition Temperatures. Langmuir 2003, 19, 7662−7664.
(38) Yoshimatsu, K.; Koide, H.; Hoshino, Y.; Shea, K. J. Preparation
of Abiotic Polymer Nanoparticles for Sequestration and Neutraliza-
tion of a Target Peptide Toxin. Nat. Protoc. 2015, 10, 595−604.
(39) Weisman, A.; Chen, Y. A.; Hoshino, Y.; Zhang, H.; Shea, K.
Engineering Nanoparticle Antitoxins Utilizing Aromatic Interactions.
Biomacromolecules 2014, 15, 3290−3295.
(40) Sundberg, L.; Porath, J. Preparation of Adsorbents for
Biospecific Affinity Chromatography: I. Attachment of Group-
Containing Ligands to Insoluble Polymers by Means of Bifunctional
Oxiranes. J. Chromatogr. A 1974, 90, 87−98.
(41) Nonlinear Least Squares Curve Fitting. http://www.colby.edu/
chemistry/PChem/scripts/lsfitpl.html (accessed Mar 13, 2021).
(42) Hoshino, Y.; Urakami, T.; Kodama, T.; Koide, H.; Oku, N.;
Okahata, Y.; Shea, K. J. Design of Synthetic Polymer Nanoparticles
That Capture and Neutralize a Toxic Peptide. Small 2009, 5, 1562−
1568.
(7) Gronemeyer, P.; Ditz, R.; Strube, J. Trends in Upstream and
Downstream Process Development for Antibody Manufacturing.
Bioengineering 2014, 1, 188−212.
(8) Tripathi, N. K.; Shrivastava, A. Recent Developments in
Bioprocessing of Recombinant Proteins: Expression Hosts and
Process Development. Front. Bioeng. Biotechnol. 2019, 7, 240.
(9) Zhao, X.; Li, G.; Liang, S. Several Affinity Tags Commonly Used
in Chromatographic Purification. J. Anal. Methods Chem. 2013, 2013,
581093.
(10) Waugh, D. S. Making the Most of Affinity Tags. Trends
Biotechnol. 2005, 23, 316−320.
(11) Young, C. L.; Britton, Z. T.; Robinson, A. S. Recombinant
Protein Expression and Purification: A Comprehensive Review of
Affinity Tags and Microbial Applications. Biotechnol. J. 2012, 7, 620−
634.
(12) Rajan, S. S.; Lackland, H.; Stein, S.; Denhardt, D. T. Presence
of an N-Terminal Polyhistidine Tag Facilitates Stable Expression of an
Otherwise Unstable N-Terminal Domain of Mouse Tissue Inhibitor
of Metalloproteinase-1 In Escherichia Coli. Protein Expression Purif.
1998, 13, 67−72.
(13) Sun, Q.-M.; Chen, L.-L.; Cao, L.; Fang, L.; Chen, C.; Hua, Z.-
C. An Improved Strategy for High-Level Production of Human
Vasostatin120−180. Biotechnol. Prog. 2005, 21, 1048−1052.
(14) Tang, W.; Sun, Z.-Y.; Pannell, R.; Gurewich, V.; Liu, J.-N. An
Efficient System for Production of Recombinant Urokinase-Type
Plasminogen Activator. Protein Expression Purif. 1997, 11, 279−283.
(15) Kou, G.; Shi, S.; Wang, H.; Tan, M.; Xue, J.; Zhang, D.; Hou,
S.; Qian, W.; Wang, S.; Dai, J.; Li, B.; Guo, Y. Preparation and
Characterization of Recombinant Protein ScFv(CD11c)-TRP2 for
Tumor Therapy from Inclusion Bodies in Escherichia Coli. Protein
Expression Purif. 2007, 52, 131−138.
(16) Mayer, A.; Sharma, S. K.; Tolner, B.; Minton, N. P.; Purdy, D.;
Amlot, P.; Tharakan, G.; Begent, R. H. J.; Chester, K. A. Modifying an
Immunogenic Epitope on a Therapeutic Protein: A Step towards an
Improved System for Antibody-Directed Enzyme Prodrug Therapy
(ADEPT). Br. J. Cancer 2004, 90, 2402−2410.
(17) Chen, H.; Xu, Z.; Xu, N.; Cen, P. Efficient Production of a
Soluble Fusion Protein Containing Human Beta-Defensin-2 in E. coli
Cell-Free System. J. Biotechnol. 2005, 115 (3), 307−315.
(18) Loughran, S. T.; Walls, D. Tagging Recombinant Proteins to
Enhance Solubility and Aid Purification. In Protein Chromatography:
Methods and Protocols; Walls, D., Loughran, S. T., Eds.; Methods Mol.
Biol.; Springer: New York, NY, 2017; pp 131−156.
(19) Dyson, M. R.; Shadbolt, S. P.; Vincent, K. J.; Perera, R. L.;
McCafferty, J. Production of Soluble Mammalian Proteins in
Escherichia Coli: Identification of Protein Features That Correlate
with Successful Expression. BMC Biotechnol. 2004, 4, 32.
(20) Hammarström, M.; Hellgren, N.; van den Berg, S.; Berglund,
H.; Härd, T. Rapid Screening for Improved Solubility of Small
Human Proteins Produced as Fusion Proteins in Escherichia Coli.
Protein Sci. 2002, 11, 313−321.
(21) Nallamsetty, S.; Waugh, D. S. Solubility-Enhancing Proteins
MBP and NusA Play a Passive Role in the Folding of Their Fusion
Partners. Protein Expression Purif. 2006, 45, 175−182.
(22) Dias, A. M. G. C.; Roque, A. C. A. The Future of Protein
Scaffolds as Affinity Reagents for Purification. Biotechnol. Bioeng.
2017, 114, 481−491.
(23) Wood, D. W. New Trends and Affinity Tag Designs for
Recombinant Protein Purification. Curr. Opin. Struct. Biol. 2014, 26,
54−61.
(43) Yoshimatsu, K.; Yamazaki, T.; Hoshino, Y.; Rose, P. E.; Epstein,
L. F.; Miranda, L. P.; Tagari, P.; Beierle, J. M.; Yonamine, Y.; Shea, K.
1704
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705

Biomacromolecules
pubs.acs.org/Biomac
Article
J. Epitope Discovery for a Synthetic Polymer Nanoparticle: A New
Strategy for Developing a Peptide Tag. J. Am. Chem. Soc. 2014, 136,
1194−1197.
(44) Culver, H. R.; Sharma, I.; Wechsler, M. E.; Anslyn, E. V.;
Peppas, N. A. Charged Poly(N-Isopropylacrylamide) Nanogels for
Use as Differential Protein Receptors in a Turbidimetric Sensor Array.
Analyst 2017, 142, 3183−3193.
(45) Panganiban, B.; Qiao, B.; Jiang, T.; DelRe, C.; Obadia, M. M.;
Nguyen, T. D.; Smith, A. A. A.; Hall, A.; Sit, I.; Crosby, M. G.;
Dennis, P. B.; Drockenmuller, E.; Olvera de la Cruz, M.; Xu, T.
Random Heteropolymers Preserve Protein Function in Foreign
Environments. Science 2018, 359 (6381), 1239−1243.
(46) Kaufmann, M.; Lindner, P.; Honegger, A.; Blank, K.; Tschopp,
M.; Capitani, G.; Pluckthun, A.; Grutter, M. G. Crystal Structure of
̈ ̈
the Anti-His Tag Antibody 3D5 Single-Chain Fragment Complexed
to Its Antigen. J. Mol. Biol. 2002, 318, 135−147.
(47) Hoshino, Y.; Ohashi, R. C.; Miura, Y. Rational Design of
Synthetic Nanoparticles with a Large Reversible Shift of Acid
Dissociation Constants: Proton Imprinting in Stimuli Responsive
Nanogel Particles. Adv. Mater. 2014, 26, 3718−3723.
(48) Hoshino, Y.; Miyoshi, T.; Nakamoto, M.; Miura, Y. Wide-
Range pKa Tuning of Proton Imprinted Nanoparticles for Reversible
Protonation of Target Molecules via Thermal Stimuli. J. Mater. Chem.
B 2017, 5, 9204−9210.
(49) Moreira, I. S.; Fernandes, P. A.; Ramos, M. J. Hot SpotsA
Review of the Protein−Protein Interface Determinant Amino-Acid
Residues. Proteins: Struct., Funct., Genet. 2007, 68, 803−812.
(50) Bogan, A. A.; Thorn, K. S. Anatomy of Hot Spots in Protein
Interfaces11Edited by J. Wells. J. Mol. Biol. 1998, 280, 1−9.
(51) Bignotti, F.; Penco, M.; Sartore, L.; Peroni, I.; Mendichi, R.;
Casolaro, M.; D’Amore, A. Synthesis, Characterisation and Solution
Behaviour of Thermo- and pH-Responsive Polymers Bearing L-
Leucine Residues in the Side Chains. Polymer 2000, 41, 8247−8256.
(52) Bauri, K.; Nandi, M.; De, P. Amino Acid-Derived Stimuli-
Responsive Polymers and Their Applications. Polym. Chem. 2018, 9,
1257−1287.
(53) Oya, T.; Enoki, T.; Grosberg, A. Y.; Masamune, S.; Sakiyama,
T.; Takeoka, Y.; Tanaka, K.; Wang, G.; Yilmaz, Y.; Feld, M. S.; Dasari,
R.; Tanaka, T. Reversible Molecular Adsorption Based on Multiple-
Point Interaction by Shrinkable Gels. Science 1999, 286, 1543−1545.
(54) Hoshino, Y.; Koide, H.; Furuya, K.; Haberaecker, W. W.; Lee,
S.-H.; Kodama, T.; Kanazawa, H.; Oku, N.; Shea, K. J. The Rational
Design of a Synthetic Polymer Nanoparticle That Neutralizes a Toxic
Peptide in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 33−38.
(55) Lindner, P.; Bauer, K.; Krebber, A.; Nieba, L.; Kremmer, E.;
Krebber, C.; Honegger, A.; Klinger, B.; Mocikat, R.; Pluckthun, A.
̈
Specific Detection of His-Tagged Proteins with Recombinant Anti-
His Tag ScFv-Phosphatase or ScFv-Phage Fusions. BioTechniques
1997, 22, 140−149.
(56) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry:
Diverse Chemical Function from a Few Good Reactions. Angew.
Chem., Int. Ed. 2001, 40, 2004−2021.
1705
https://doi.org/10.1021/acs.biomac.1c00119
Biomacromolecules 2021, 22, 1695−1705