C.-T. Chen et al.
added. The solution was heated to 408C for at least 1 h; then TEOS
(8.5 g) was added. The resulting solution was stirred for 20 h at 408C,
and then the mixture was aged at 1008C for 24 h. After filtration and
washing, the solid was dried at 408C and then calcined at 5608C for 6 h.
The resulting OMPS material is denoted as MCF.
88%). Repeating the above deprotection and coupling reactions pro-
duced the his tags with the desired lengths. l-CysACHTNUTRGNEUNG(Trt)-OH was added to
the N-terminus of the histidine tags for reacting with maleimide. The
trityl groups were removed by dilute acid.
Chemical modification of histidine tags with different lengths to HRP
and immobilization to MCF-NTA: HRP was modified with a cross heter-
obifunctional crosslinker according to the procedure recommended by
the commercial supplier. N-(3-Maleimidopropionyloxy)succinimide ester
or N-(e-maleimidocarproyloxy)succinimide ester (3 equiv) was added to
a solution containing HRP (5 mg) in PBS (100 mm, pH 7.0, 1 mL) as the
crosslinker. The resulting solution was stirred at 48C for 2 h, followed by
the addition of a solution containing His6, His4, His3, or His2 (10 equiv)
bearing a cysteine residue for the reaction with maleimide in PBS
(100 mm, containing 0.1m ethylenediaminetetraacetic acid (EDTA),
1 mL) at pH 7.0. The solution was then stirred at 48C for 4 h. The bio-
conjugates were purified by gel filtration (PD-10 desalting column) to
remove excess crosslinker and EDTA and unreacted histidine tags. His6-,
His4-, or His3-tagged HRP (0.2 mL, 1 mgmLÀ1) was incubated with
MCF-NTA (1 mg), which was suspended in PBS (10 mm, 1 mL) at pH 7.0
first, for 6 h at 48C. The binding affinity was evaluated by MALDI-TOF
MS in combination with SDS-PAGE, similar to the procedures used for
the recombinant UPPs.
Grafting NTA to the surface of the OMPSs and nickelated NTA-
OMPSs: Two different approaches, direct silanization and stepwise chem-
ical modification, were employed to fabricate NTA-H2O, and dried in an
oven at 708C. To form chelated Ni-NTA, each NTA-OMPS was mixed
with NiCl2 (10 mm). Typically, NTA-OMPS (1 mg) was mixed with a solu-
tion of NiCl2 (1.5 mL, 10 mm) with stirring at room temperature for 6 h.
The resulting materials were then collected by centrifugation, washed
with phosphate buffered saline, and used directly.
OMPS and NTA-OMPS characterization: N2 adsorption–desorption iso-
therms were measured by using a Micromeritics Tristar 3000 instrument
at liquid nitrogen temperature (77 K). Before the measurements, the
samples were degassed at 1208C for 12 h. The surface area was measured
by using the BET method.[49] The pore size data were analyzed by the
Barrett–Joyner–Halenda (BJH) method on the adsorption branches of
the N2 isotherm. XRD patterns were obtained on a PANalytical X’Pert
Pro diffractometer by using CuKa radiation (l=1.5418 ꢃ) at 45 kV and
40 mA. The data were collected from 0.5–58 (2q). Thermogravimetric
analyses were conducted on a Dynamic TGA 2950 TG analyzer with a
heating speed of 108CminÀ1 under air. Elemental analyses were per-
formed on a Heraeus CHNS elemental analyzer. TEM was performed on
a Hitachi H-7100 electron microscope operating at 75 kV.
Immobilization of His6-tagged HRP in NTA-OMPSs for catalysis: The
His6-tagged HRP was immobilized in NTA-OMPSs as follows: The ini-
tial protein concentrations in solution were calculated from the absorb-
ance at l=403 nm (heme protein, e=100000mÀ1 cmÀ1) for His-tagged
HRP in PBS (10 mm) at pH 7.0. Each NTA-OMPS (1–5 mg) was placed
in the protein solution at 48C for 14 h, followed by centrifugation at
9000 rpm for 5 min. The UV/Vis absorbance of the supernatant at l=
403 nm was measured again to calculate the concentration of the remain-
ing protein in solution. The amount of protein immobilized in the NTA-
OMPS was estimated by the difference in the protein concentrations
before and after immobilization.
Purification of histidine-tagged proteins by NTA-OMPSs: UPPs ex-
pressed from a pET32a vector bearing a His tag was used for the purifi-
cation experiment.[47,55] NTA-OMPS (5 mg) was incubated in PBS (1 mL,
10 mm) at pH 7.0, and then cell lysate (0.1 mL) was mixed with the NTA-
OMPS for 6 h at 48C. The NTA-OMPS was separated from the unbound
UPPs by centrifugation at 9000 rpm for 5 min. After the supernatant was
removed, the NTA-OMPS was washed three times with PBS (100 mm,
containing 1.38m NaCl) to remove other non-specific adsorbed proteins.
The UPPs immobilized in the Ni-NTA-OMPS could be eluted from the
Ni-NTA-OMPS by adding OMPSs (500 mm). In direct silanization, the
NTA-silane was grafted to the OMPSs to yield MCF-NTA, film-NTA,
and SBA-NTA. Usually NTA-silane (200 mg) was added to a suspension
of OMPS (400 mg) in dry toluene (40 mL) and stirred at 258C for 2 h
and then heated to 1108C. After 18 h, the resulting solution was concen-
trated by evaporation to give a white powder. The unreacted organosi-
lane was removed by washing the resulting solid with EtOH, and then
the solid was dried in an oven at 708C. Subsequently, the material was
hydrolyzed at pH 2.0 for 6 h to remove the methyl ester groups. Stepwise
chemical modification involved first grafting MPTES onto the surface of
the OMPSs by using the procedure described above, followed by Michael
addition of NTA-maleimide in a phosphate buffer (100 mm) at pH 7.0 for
6 h to afford MCF-S-NTA, film-S-NTA, and SBA-S-NTA. The resulting
white solid was filtered, washed with imidazole (100 mm in PBS at
pH 7.0). SDS-PAGE and MALDI-TOF MS (Bruker Biflex) were em-
ployed to analyze the His-tagged protein (mixed with matrix sinapinic
acid in 0.01% trifluoroacetic acid and 50% CH3CN).
Enzyme leakage, activity assay, and selective hydroxylation of l-tyrosine
to l-DOPA catalyzed HRP: To assess the effect of the pH on the
enzyme leakage, His-tagged HRP immobilized in NTA-OMPS was
placed in PBS (100 mm) at pH 7.0, 6.0, 5.0, or 4.0 for 24 h at 48C. The
amount of leaching was evaluated by measuring the absorbance at l=
403 nm. The activity of immobilized His-tagged HRP was determined
spectrophotometrically by using ABTS as the electron donor and H2O2
as the substrate.[58] The increase in the absorbance of the ABTS radical
at l=405 nm (e=36800mÀ1 cmÀ1) with the time was monitored at 258C.
Each sample contained OMPS (1 mg, 0.176 mg His-tagged HRP per g
SiO2), ABTS (2 mm), and H2O2 (3.0 mm) in potassium phosphate buffer
(100 mm, 3 mL, pH 5.0). One unit of enzyme is defined by the oxidization
of 1 mmol of ABTS per minute at pH 5.0 and 258C.
Selective hydroxylation of l-tyrosine to l-DOPA was conducted by a pro-
cedure similar to that reported in the literature.[59] A solution of l-tyro-
sine (2 mm, 500 mL) in acetate buffer (60 mm, pH 5.0) was placed in a
round-bottom flask. Dihydroxyfumaric acid (2 mmol) and immobilized
HRP (0.25 mg) were added to the solution and O2 was bubbled through
the solution at 08C with vigorous stirring. After the first hour, additional
dihydroxyfumaric acid (2 mmol) was added to the reaction, which was
terminated by acidification after 3 h from the beginning of the reaction
to avoid over-hydroxylation of the product. The resulting mixtures were
first run through a column packed with Dowex-50-4X (cationic form,
200–400 mesh) resins eluted with HCl (0.7n). The collected fractions
were lyophilized, and the residue was purified to obtain l-DOPA by C8
reverse-phase high-performance liquid chromatography (HPLC). The re-
action conversion was calculated on the basis of the HPLC traces.
Synthesis of histidine tags with different lengths (His6, His4, and His3):
Solution-phase peptide synthesis was adopted to synthesize His tags with
different lengths. Structural characterizations of these peptides are avail-
able in the Supporting Information. The general protocol is as follows: A
solution of piperidine in DMF (20%, 10 mL) was added to the commer-
cially available Trt- and Fmoc-protected histidine ester [Fmoc-HisACTHNUTRGNEU(GN Trt)-
OMe] (1 mmol). The resulting solution was stirred at ambient tempera-
ture for 30 min. After removal of excess solvent, the residue was purified
by flash column chromatography by eluting with CH2Cl2/CH3OH (100:4
Thermal stability: Thermal stability experiments were conducted by heat-
ing His-tagged HRP immobilized in Ni-NTA-OMPSs at 708C for 30, 60,
or 90 min. The enzymatic activity was determined by the degree of
phenol degradation.[19,20,60] Each sample contained phenol (4 mL,
5000 ppm), H2O2 (1 mL, 30%), Tris buffer (50 mm, 200 mL, pH 7.5), and
OMPS (1 mg, the enzyme loading was about 0.176 mg His-tagged HRP
per g SiO2) for polymerization at 708C for 30, 60, or 90 min. After centri-
fugation, an aliquot of the supernatant (100 mL) was mixed with potassi-
v/v) to give
(0.77 mmol), Fmoc-His
yl]-3-ethyl-carbodiimide hydrochloride (1.0 mmol), and
a
yellow oil of His
(Trt)-OH (0.9 mmol), 1-[3-(dimethylamino)prop-
catalytic
ACHUTNGTRENNUG(Trt)-OMe (81%). HisACHTUNGTNER(NUGN Trt)-OMe
ACHTUNGTRENNUNG
a
amount of HOBt (50 mg) were dissolved in CH2Cl2 (10 mL) and the solu-
tion was stirred at room temperature for 12 h. Removal of excess solvent
was followed by purification by flash column chromatography by eluting
with CH2Cl2/CH3OH (100:3 v/v) to afford an off-white solid (0.68 mmol,
13066
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 13059 – 13067
Lin, Yu-Chung
