Beilstein J. Org. Chem. 2015, 11, 784–791.
calcium (HBS-Ca), 20 mM HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid), pH 7.4, 150 mM NaCl, 1 mM
For subsequent dual-functionalization, the samples were CaCl2 were injected over both lanes at a flow rate of 30 µL/min.
centrifuge-filtered with Dulbecco’s PBS buffer (100 mM, pH 7) The final binding signals were obtained by subtracting the
after oxime ligation and directly applied in the CuAAC.
resulting response units (RU) of the free reference lane from the
10 µM; 100 mM phosphate buffer, 100 mM NaCl, pH 7) was lowed by a 180 s dissociation phase. Washing and regenerating
(
mixed with CuSO4 (1 M in 100 mM phosphate buffer, 100 mM of both lanes was done by injecting 4 M MgCl2.
NaCl, pH 7), sodium ascorbate (50 equiv to Cu2+) and 1-O-but-
3
-ynyl-α-galactopyranoside (2) (1100 equiv to protein), 80 µL For KD determination, chips were loaded to one third with the
THPTA (5 equiv to Cu2+), and aminoguanidine (8 mM) and respective TTL and 50 µL ECL were injected in each run with a
tions were centrifuge-filtered (14000 r/min) and washed 3× with reference flow cell. Washing and regeneration was done again
buffer/EDTA-solution (100 mM phosphate buffer, 100 mM by injecting 4 M MgCl2. Kinetic measurements consisted of at
NaCl, 5 mM EDTA, pH 7) and 4× with ultrapure water. The least five different concentrations ECL (1, 2, 10, 20 and
(
Coomassie stain) and Western blotting (streptavidin–peroxi- Gal-3), KDs were determined twice. For the TTL without galac-
BioRad) (see Figure 1). Protein concentrations were checked possible lectin concentration (100 µM). Data were aligned and
(
by UV (λ = 280 nm).
Lipase activity test [53]. Lipase activity was determined by analyzed on equilibrium binding by nonlinear curve fitting of
ethanol) and solution B (100 mg gummi arabicum in 90 mL
Tris-HCl buffer (50 mM, pH 8)) were mixed 1:9 and dispersed
Supporting Information
(
ultraturrax, 3 min, 20000 min−1) to get solution C. For each
measurement, 450 µL of solution C were mixed with 50 µL
enzyme solution (0.13 nmol protein). The contribution of auto-
hydrolysis was assessed by including a blank that contained the
same volume of 50 mM Tris·HCl pH 8.0 instead of enzyme
Details on materials, protein design, construction of the
expression plasmids, protein expression and purification,
mass spectrometry data for the expressed proteins, general
methods, synthetic protocols and analytical data (including
(
background measurement). The samples were shaken at 50 °C
1
H, 13C and 19F NMR spectra) for compounds 1 and 2,
reaction conditions for the ligation strategies, SDS PAGE
and Western Blot lanes are provided as Supporting
Information.
Surface-plasmon-resonance (SPR). SPR measurements were
performed on a BiacoreX (GE Healthcare, Freiburg, Germany).
Biotinylated TTL samples were coupled to streptavidin func-
tionalized gold chips (SA-Chips, GE Healthcare, Freiburg,
Germany). Before immobilization, the sensor chip was condi-
tioned with three consecutive 1 min injections of 1 M NaCl and
Supporting Information File 1
5
0 mM NaOH.
Acknowledgements
For initial binding experiments, flow cell 2 (Fc2) of each chip The authors acknowledge support from the DFG (SFB 765 and
was fully loaded (≈400 RU) with our protein. Flow cell 1 (Fc1) SPP 1623), the BMBF (Biokatalyse 2021), the Fonds der
remained untreated and served as a reference. After immobili- Chemischen Industrie (FCI), the Einstein Foundation, the
zation, a sample volume of 100 µL of different concentrations Boehringer-Ingelheim Foundation (Plus 3 award) and the Studi-
789