Chaozhan Wang

High Recovery Refolding of rhG‐CSF from Escherichia coli, Using Urea Gradient Size Exclusion Chromatography

Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) expressed in Escheriachia coli ( E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG‐CSF in 8.0 mol·L‐1 urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG‐CSF molecules was decreased linearly, and the denatured rhG‐CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG‐CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG‐CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG‐CSF refolding&‐;in terms of specific bioactivity and mass recovery, the denatured rhG‐CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 μL of solubilized and denatured rhG‐CSF in 8.0 mol·L‐1 urea solution with a total protein concentration of 2.3 mg·mL‐1 was loaded onto the SEC column, rhG‐CSF with a specific bioactivity of 1.0 × 108 IU·mg‐1 was obtained, and the mass recovery was 46.1%.

The preparation of high-capacity boronate affinity adsorbents by surface initiated reversible addition fragmentation chain transfer polymerization for the enrichment of ribonucleosides in serum.

Boronate affinity adsorption is uniquely selective for cis-diol-containing molecules. The preparation and application of boronate affinity materials has attracted much attention in recent years. In this work, a high-capacity boronate affinity adsorbent was prepared by surface-initiated reversible addition-fragmentation chain transfer polymerization (SI-RAFT). Commercial aminated poly(glycidyl methacrylate) (PGMA) microspheres were modified with the chain transfer agent (CTA) S-1-dodecyl-S-(α,α-dimethyl-α-acetic acid)trithiocarbonate (DDATC). Boronate-affinity adsorbents were then prepared via SI-RAFT polymerization employing 3-acrylamidophenylboronic acid (AAPBA) as the monomer. The Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption and desorption measurements have proven the successful grafting of AAPBA on PGMA microspheres surface. The boronate affinity adsorbents thus prepared possess much higher adsorption capacity (99.2 µmol/g of adenosine) and both faster adsorption and desorption speed towards ribonucleosides, the adsorption and desorption could be completed in 2 min. The high selectivity of the adsorbents to ribonucleosides was verified in the presence of a large excess of deoxynucleosides. The boronate affinity adsorbents were then employed for sample pretreatment before HPLC analysis of ribonucleosides in serum. The ribonucleosides were effectively enriched by boronate affinity dispersive solid-phase extraction (BA-DSPE), with high mass recoveries and good precision. The simultaneous determination of uridine and guanosine in calf serum was achieved by utilizing the standard addition method, their contents were determined to be 170 ± 11.6 ng/mL and 39.6 ± 4.4 ng/mL respectively. The results proved that the prepared boronate affinity materials could be applied for sample pretreatment of cis-diol containing molecules in biological samples.

Renaturation of Recombinant Human Granulocyte Colony‐Stimulating Factor Produced from Escherichia coli Using Size Exclusion Chromatography

Abstract Refolding with simultaneously partial purification of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) expressed in Escherichia coli (E. coli) by size exclusion chromatography (SEC) is presented in this work. The solution containing the denatured and reduced rhG‐CSF in 8.0 mol · L−1 urea extracted from the inclusion body was directly injected into a Superdex 75 column and the refolded rhG‐CSF was obtained after elution from the column. Several factors, including the concentration of urea in the mobile phase, pH, flow rate, concentration of glutathione, and ratio of GSH to GSSG, concentration of glycerol, sample loading volume, effecting the aim protein refolding were investigated in details. With the selected optimal conditions, the denatured and reduced rhG‐CSF was successfully refolded by SEC, and was partially purified during the chromatographic process. When 200 µL of denatured rhG‐CSF at a concentration of 2.3 mg · mL−1 was loaded on the SEC column, rhG‐CSF with specific activity of 1.2×108 IU · mg−1, purity of 83%, and mass recovery of 30% was obtained.

Preparation of an internal surface reversed-phase restricted-access material for the analysis of hydrophobic molecules in biological matrices

Restricted-access materials (RAMs) have been broadly used for sample pretreatment in the chromatographic analysis of biological samples. In the present work, a novel internal surface reversed-phase (ISRP) RAM was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). Octadecyl and 4-(chloromethyl)phenyl were immobilized on silica using a one-pot synthesis approach to form a reversed-phase layer to retain small hydrophobic molecules, allowing the modified silica to serve as a macro-initiator. Then, poly(glycidyl methacrylate) (pGMA) was grafted onto the surface via SI-ATRP, and the epoxy groups were further hydrolyzed to form an external hydrophilic layer. The properties of this ISRP-RAM for the retention of small molecules and the exclusion of proteins were evaluated using solid-phase extraction (SPE). The removal efficiencies of bovine serum albumin (BSA) and lysozyme were 94.9% and 93.5%, respectively. The recoveries of five drugs, puerarin, p-hydroxybenzaldehyde, loratadine, nifedipine and diazepam, were 93.2-116%. Furthermore, the ISRP-RAM was employed for the SPE of five phthalate esters (PAEs) from bovine milk prior to high-performance liquid chromatography (HPLC) analysis. The results indicate that the prepared ISRP-RAM is qualified for practical bioanalysis.

Refolding of Denatured/Reduced Lysozyme at High Concentrations by Artificial Molecular Chaperone-Ion Exchange Chromatography

Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone‐ion exchange chromatography (AMC‐IEC). This new method is applied to the refolding of a widely used model protein, urea‐denatured/dithiothreitol‐reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, β‐cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC‐IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC‐IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL−1, the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC‐IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC‐IEC is a high efficient and low cost protein refolding method.

Expression, refolding, and purification of a truncated human Delta-like1, a ligand of Notch receptors

The Notch signaling pathway plays a pivotal role in proliferation, apoptosis, and cell fate specification in both embryonic and postnatal development, and is a potential therapeutic target for human diseases such as cancer. To express in Escherichia coli and purify soluble fragment of human Delta-like1 (hDll1), we cloned two extracellular fragments of hDll1 [hDll1 (127-225) and hDll1 (26-225)]. The hDll1 (127-225) fragment was successfully expressed in E. coli as a GST fusion protein (GST-hDll1). The GST-hDll1 protein, which was expressed as inclusion bodies after induction by IPTG, was refolded and purified simultaneously using affinity chromatography and size exclusion chromatography. The purified GST-hDll1 was of more than 95% purity, and had a molecular weight of 39kDa. Reporter assay showed that GST-hDll1 could activate a reporter gene that is dependent on Notch activation. Therefore, using the E. coli expression system and different chromatography systems, we successfully expressed, refolded, and purified a biologically active GST-hDll1, which might be potentially useful for therapy and studying the Notch pathway.

Zirconium-doped magnetic microspheres for the selective enrichment of cis-diol-containing ribonucleosides

Zirconium-doped magnetic microspheres (Zr-Fe3O4) for the selective enrichment of cis-diol-containing biomolecules were easily synthesized via a one-step hydrothermal method. Characterization of the microspheres revealed that zirconium was successfully doped into the lattice of Fe3O4 at a doping level of 4.0 at%. Zr-Fe3O4 possessed good magnetic properties and high specificity towards cis-diol molecules, as shown using 28 compounds. For ribonucleosides, the adsorbent not only has favorable anti-interferential abilities but also has a high adsorption capacity up to 159.4μmol/g. As an example of a real application, four ribonucleosides in urine were efficiently enriched and detected via magnetic solid-phase extraction coupled with high-performance liquid chromatography. Under the optimized extraction conditions, the detection limits were determined to be between 0.005 and 0.017μg/mL, and the linearities ranged from 0.02 to 5.00μg/mL (R≥0.996) for these analytes. The accuracy of the analytical method was examined by studying the relative recoveries of the analytes in real urine samples, with recoveries varying from 77.8% to 119.6% (RSDs<10.6%, n=6). The results indicate that Zr-Fe3O4 is a suitable adsorbent for the analysis of cis-diol-containing biomolecules in practical applications.

Synthesis and application of boronic acid‐functionalized magnetic adsorbent for sensitive analysis of salbutamol residues in pig tissues

Salbutamol (SAL) is the most widely used β2 -agonist drug for asthma and chronic obstructive pulmonary patients, but it is also often abused as feed additive. In recent years, the abuse of SAL has led to a large number of food safety incidents. Therefore, the monitoring of SAL residues in animal products is very important. A highly selective boronate affinity magnetic adsorbent was synthesized and developed for detection of trace levels of SAL residues in pig tissue samples. The obtained Fe3O4@SiO2@FPBA(4-formylphenylboronic acid) magnetic adsorbent showed good adsorption ability to catechol and SAL, and then it was successfully applied as special magnetic solid-phase phase extraction adsorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous isolation and determination of cis-diol compounds. The binding capacity of catechol and SAL reached 96 and 50 µmol/g, respectively. The method was successfully established for the detection of trace levels of SAL in pig tissue samples. The linear range extended from 0.32 to 800 µg/kg (R(2) = 0.9994). The limit of detection of SAL was 0.19 µg/kg. The recoveries were satisfactory (89.5-108.0%) at three spiked levels with RSD between 2.1 and 11.3%. These results indicated that the method has potential for enrichment and detection of trace levels of SAL residual in animal food products.

Preparation of hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase with adjustable selectivity by controlling different ratios of the co-monomers

Development of mixed-mode chromatography (MMC) stationary phase with adjustable selectivity is beneficial to meet the needs of complex samples. In this work, surface-initiated atom transfer radical polymerization (SI-ATRP) using the mixture of two functional monomers was proposed as a new preparation strategy for MMC stationary phase with adjustable selectivity. The mixture of sodium 4-styrenesulfonate (NASS) and dimethylaminoethyl methacrylate (DMAEMA) underwent SI-ATRP to bond poly(NASS-co-DMAEMA) on the surface of silica to prepare hydrophilic interaction/ion-exchange mixed-mode stationary phase. Various analytes (neutral, acidic, basic analytes and strong polar nucleosides) were employed to investigate the retention behaviors. The influences of water content and pH of the mobile phase on the retention validated the mixed-mode retention mechanisms of HILIC and ion-exchange. The charge and polarity of stationary phase as well as the separation selectivity were conveniently manipulated by the ratio of NASS to DMAEMA monomer, and the use of DMAEMA in the mixture additionally endowed the column with the temperature-responsive characteristics. Moreover, the application of the developed column was demonstrated by the successful separation of nucleosides, β-agonists and safflower injection. In a word, the proposed strategy can be potentially applied in the controllable preparation of MMC stationary phase with adjustable selectivity.

Preparation and evaluation of diblock copolymer‐grafted silica by sequential surface initiated‐atom transfer radical polymerization for reverse‐phase/ ion‐exchange mixed‐mode chromatography

A novel approach that involved the grafting of diblock copolymer with two types of monomer onto substrate by sequential surface initiated-atom transfer radical polymerization was proposed to prepare a mixed-mode chromatographic stationary phase. The distinguishing feature of this method is that it can be applied in the preparation of various mixed-mode stationary phases. In this study, a new reverse-phase/ion-exchange stationary phase was prepared by grafting hydrophobic styrene and cationic sodium 4-styrenesulfonate by the proposed approach onto silica surface. The chromatographic properties of the prepared stationary phase were evaluated by the separation of benzene derivatives, anilines, and β-agonists, and by the effect of pH values and acetonitrile content on the retention. Compared with typical RP columns, the prepared stationary phase achieved the better resolution and higher selectivity at a shorter separation time and lower organic content. Moreover, the application of the prepared column was proved by separating widely distributed polar and charged compounds simultaneously.