Zhaoyang Ding

Application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer.

An endo-glucanase could be efficiently purified using metal chelate affinity precipitation by a pH-response polymer PMMDN with iminodiacetic acid (IDA) and Cu(2+) as affinity ligand. In this study, docking method was used to identify the appropriate chelator and the metal ion as ligand by Grid score. The simulation results were compared with the label-free detection data analyzed by ForteBios Octet. The ligand IDA-Cu(2+) was the final choice. A pH-response polymer PMMDN was polymerized and subsequently coupled with IDA-Cu(2+)as the ligand. The pI and recovery of PMMDN and PMMDN-IDA-Cu(2+) were 4.50, 99.8% and 4.39, 97.6%, respectively. Optimal adsorption conditions were found to be ligand density of 3.0 mmol/g, pH 5.5 and 1.0 mol/L NaCl. The adsorption isotherm showed the maximum adsorption as 57.62 mg/g polymer and the dissociation constant as 1.08 mg/mL. For the elution of the PMMDN-IDA-Cu(2+) with the protein, 0.5 mol/L imidazole containing 1.0 mol/L guanidine hydrochloride was used as the eluent. Under these conditions, electrophoretic purity of endo-glucanase was obtained by only one step, and the elution recoveries were 96.45% (protein) and 93.24% (activity).

Lipase purification by affinity precipitation with a thermo-responsive polymer immobilized Cibacron Blue F3GA ligand

A thermo-responsive polymer (PNNB) was synthesized with lower critical solution temperature 27.5°C and over 95% recovery. The adsorption of porcine pancreatic lipase on Cibacron Blue F3GA-conjugated PNNB (PNNB-CB) closely followed the bi-Langmuir adsorption isotherm. The maximum adsorption capacity was found at pH 5.0, with a ligand density of 18.4 μmol/g polymers. The optimized eluent was a 0.01 M phosphate buffer solution at pH 8.0 containing 20% ethylene glycol. Six adsorptiondesorption recycles indicated excellent reusability of the affinity adsorbent. PNNB-CB was applied to separate porcine pancreatic lipase from its crude material giving a lipase activity recovery of 81.6% with a 16-fold purification factor. Lipase could be purified to single-band purity, according to gel electrophoresis. The purification strategy is therefore feasible and efficient for purifying proteins of interest.

Construction of Thermo-Responsive Elastin-Like Polypeptides (ELPs)-Aggregation-Induced-Emission (AIE) Conjugates for Temperature Sensing

In this work, an aggregation-induced emission (AIE) molecule (tetraphenylethene derivative, TPE-COOH) was conjugated to elastin-like polypeptides (ELPs40) via an amide bond to form ELPs40-TPE. The successful synthesis of ELPs40-TPE was confirmed by Circular Dichroism spectroscopy, gel electrophoresis, UV-vis absorption, and fluorescence emission spectroscopy. ELPs40-TPE possessed both amphiphilicity and the features of an AIE, and the fluorescence intensity was dependent on the local temperature. The Hela cells imaging indicated that ELPs40-TPE has great potential for bio-imaging applications because of its advantages of high fluorescence intensity, good water-solubility, and remarkable biocompatibility.

Preparation and Characterization of a pH-responsive Polymer that Interacts with Microbial Transglutaminase during Affinity Precipitation

Microbial transglutaminase (MTG) has been widely used in the food and pharmaceuticals industries. In this study, MTG was purified using affinity precipitation with an affinity polymer (PMMDN-T), which was synthesized using a pH-responsive polymer (PMMDN) coupled with L-thyroxin as an affinity ligand. Interactions between MTG and PMMDN-T were investigated using turbidimetric titration, zeta potential measurements, and low-field nuclear magnetic resonance (LF-NMR). We found different behaviors, architectures, and phase states of pH-dependent interactions between MTG and PMMDN-T interactions. Binding energetics between MTG and PMMDN-T were determined by isothermal titration calorimetry (ITC). The isoelectric point (pI) of the affinity polymer was 4.65 and was recovered with 96.7% efficiency after recycling the polymer three times. The optimal adsorption condition was 0.02 mol/L phosphate buffer (pH 6.0) with 1.0 mol/L NaCl at 30.0°C and a ligand density of 50.0 μmol/g. The maximum elution recoveries of total MTG were 98.44% (protein) with 92.19% (activity) using 0.02 mol/L pH 10.0 Gly-NaOH as the eluent.

Energy-Transfer Metal–Organic Nanoprobe for Ratiometric Sensing with Dual Response to Peroxynitrite and Hypochlorite

An energy-transfer metal–organic nanoprobe is designed for ratiometric sensing with dual response to both peroxynitrite (ONOO–) and hypochlorite (ClO–). Here, a nanoscale metal–organic framework (NMOF) acts as the energy donor and molecular probe as the acceptor to construct a Förster resonance energy transfer (FRET) nanosystem. Biocompatible dextran conveniently binds to the NMOF surface through multiple weak coordination interactions to improve water dispersibility and cell uptake. Dextran can also coordinate with the molecular probe with arylboronic acid group, which enables the convenient grafting of molecular probes to the NMOF surface to construct energy-transfer nanoprobes. Because of efficient FRET, the bright blue fluorescence of NMOF is quenched, whereas red emission from the acceptor is enhanced. Upon reacting with ONOO–, the probe departs from NMOF and the fluorescence of NMOF is recovered because of the interruption of FRET. When reacting with ClO–, the phenothiazine moiety in the molecular probe is oxidized into phenothiazine-5-oxide, which leads to more efficient energy transfer and the fluorescence shifts from red to orange. The nanoprobes are also successfully applied to the detection of ONOO– and ClO– in living cells.