Yongdong Huang

Chinese Academy of Sciences, Peping, Beijing, China

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Publications (24)50.78 Total impact

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    ABSTRACT: Some microorganisms can selectively capture carbon dioxide without light irradiation, which proposes a wide application prospect. The purpose of this study was to create a microorganism-nanoparticle assembly, which will be used for carbon dioxide fixation and in situ conversion into a platform chemical, succinic acid. Firstly, uniform size-controlled magnetic nanomaterial were synthesized and well assembled with non-photosynthetic carbon-fixation microorganism Actinobacillus succinogenes 130Z. CO2 capture efficiency can be improved dramatically by enhancing the transfer of carbon dioxide between gas phase and cytoplasm by the hydrophilic oleate-modified Fe3O4 nanoparticles. The assembly will integrate the advantages of two processes, carbon dioxide sequestration by cells and carbon dioxide adsorption by chemical reagents, which resulted in 71 mmol CO2 fixation/g dry cell in 24 h. This article provides a basic study for CO2 sequestration and carbon resource utilization.
    No preview · Article · Mar 2016 · International Journal of CO2 Utilization
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    ABSTRACT: Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamics properties were improved greatly after the dextran-grafting process and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2016 · Journal of Separation Science
  • Rongyue Zhang · Qiang Li · Dekun Ji · Yiting Pan · Bo Xu · Yongdong Huang · Lan Zhao
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    ABSTRACT: Abstract The polymer monolith for solid-phase synthesis with high efficiency was prepared through in situ copolymerization of chloromethylstyrene and ethylene glycol dimethacrylate (PCMS-EDMA). The obtained monolith was grafted by two kinds of poly(ethylene glycol) acrylate oligomer, poly(ethylene glycol) acrylate (PEGA) and poly(ethylene glycol) methyl ether acrylate (mPEGA). The monolith was grafted via activators generated by electron transfer atom transfer radical polymerization (AGET ATRP) with the increased number of functional groups (-OH). About 0.61-0.81 mmol/g hydroxyl group resulted from side groups in each grafting polymer chain. PmPEGA in the grafting block copolymer chains can increase the distance between the adjacent reactive sites of PEGA (-OH) in each polymer chain. Therefore, the grafted monoliths with the block copolymer of PEGA-co-mPEGA can give high yield (85%) and purity (93%) of the crude peptide (a difficult sequence-acyl carrier protein fragment 65-74) under the condition of high loading capacity (0.76 mmol/g). These results were higher than those by the grafted monolith with only polymer of PEGA (72% and 81%, respectively) and commercial Wang resin (43% and 39%, respectively). The synthetic efficiency on the grafted monolith with block copolymer in the continuous flow technique was 5-6 folds higher than Wang resins in the manual operation conditions.
    No preview · Article · Aug 2015 · Reactive and Functional Polymers
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    Yu Lin · Yongdong Huang · Wei Zheng · Kui Wu · Qun Luo · Yao Zhao · Shaoxiang Xiong · Fuyi Wang
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    ABSTRACT: Electrospray ionization mass spectrometry (ESI-MS) has been widely used to identify binding sites of metal complexes to proteins. However, the MS quantification of the metal-protein coordination remains a challenge. We have recently demonstrated by ESI-MS analysis that organometallic ruthenium complexes [(η(6)-arene)Ru(en)Cl](+) (arene=p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en=ethylenediamine) bound to human glutathione-S-transferase π (GSTπ) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GSTπ dimer, showing inhibitory potency against the enzyme (J. Inorg. Biochem., 128 (2013) 77-84). Herein, we developed a mass spectrometric method to quantify the binding stoichiometry of the three complexes to GSTπ. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GSTπ complexes and the respective light-labelled peptides in the tryptic digest of equimolar GSTπ were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GSTπ with its substrate GSH significantly reduced the bindings of the ruthenium complexes at Met92 and Cys102, but had little impact on the bindings at Cys15 and Cys48. As the inhibitory activities of the ruthenium complexes against GSTπ are similar to those against GSTπ in complexation with GSH, these results suggest that the inhibition of the ruthenium complexes on GSTπ is attributed to the ruthenation at Cys15 and Cys48. The present work provides not only insights into the understanding on the inhibitory mechanism of ruthenium complexes GSTπ, but also a general method for quantitative characterization of metal-protein interactions. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015 · Journal of Inorganic Biochemistry
  • Rongyue Zhang · Qiang Li · Yongdong Huang · Lan Zhao · Peili Ye · Guanghui Ma · Zhiguo Su
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    ABSTRACT: The polymer monolith for solid-phase synthesis was prepared through in situ copolymerization of chloromethylstyrene and ethylene glycol dimethacrylate (PCMS-EDMA), and the obtained monolith was grafted by poly (ethylene glycol) acrylate monomer via activators generated by electron transfer atom transfer radical polymerization (AGET ATRP). The novel monolith was highly crosslinked and showed no detectable swelling in both polar and nonpolar solvents (e.g. dichloromethane, dimethylformamide, tetrahydrofuran, acetonitrile, and methanol). The grafted monolith increased the number of functional groups in the range of 0.32-0.85 mmol/g, which resulted from side groups in each grafting polymer chain. Meanwhile, the grafted monolith showed good permeability and mechanical strength under the flow-through conditions. This monolith was derived into Wang resin and used in the synthesis of a difficult sequence-acyl carrier protein fragment 65-74 (ACP 65-74) in a new designed continuous flow equipment. The yield and purity of the crude peptide acquired from the grafted monolith reached 82% and 92%, respectively, which were higher than those by the ungrafted monolith (51% and 60%, respectively) or commercial Wang resin (51% and 61%, respectively). The synthetic efficiency on the grafted monolith in the continuous flow technique was 4-5 folds higher than on Wang resins in the manually operation conditions. Therefore, this monolithic resin showed potential effects in production scale-up.
    No preview · Article · Feb 2015 · Polymer
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    ABSTRACT: Antithrombin III (AT III) is the most important anti-clotting substance. Recombinant human antithrombin III (rhAT III) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT III is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT III by isoelectric precipitation and heparin affinity chromatography. First, casein was effectively removed by isoelectric precipitation. rhAT III was further purified by heparin affinity chromatography. In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT III were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied. Under the optimized conditions, the protein recovery of rhAT III was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process is very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.
    No preview · Article · Oct 2014 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
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    ABSTRACT: High hydrophilicity of gigaporous microspheres based on a copolymer of poly(glycidyl methacrylate)-co-divinyl benzene (PGMA-DVB) was successfully realized through coating the branched polyethyleneimine (PEI) in PGMA-DVB microspheres. PEI with various molecules weights and different branching agents were identified in terms of protein recovery as evaluation approach. For this evaluation, PEI600 (Mw=600) and poly (ethylene glycol) diglycidyl ether (PEGDE, Mw=400) were used as modification agent and branching agent, respectively. The modified microspheres showed good permeability and revealed a certain mechanical strength. After modification, the protein recovery increased from 40% to >90%. The protein recovery increased with the branched generations and the first and second generations could give the protein recovery of 93% and 96%, respectively. Meanwhile, the ionic capacity also showed a rising trend in the range of 0.11-0.32mmol/mL with the branched generations. But the dynamic binding capacity of protein (bovine serum albumin, BSA as the model protein) increased at first and then decreased. Analysis of the dry microspheres structure by mercury intrusion method as well as observation of the branched PEI on PGMA-DVB membrane in aqueous solution indicated that excess PEI chains with the extended state in the second generation would block the small pores and decrease the accessible surface area. Therefore, the protein capacity on the second generation, on the contrary, was lower than that on the first generation. Meanwhile, it was found that the PEI chains in the modified microspheres changed their construction from the extended to the collapsed state with increase of NaCl concentration. And the corresponding pore size of the modified microspheres increased with salt concentration through low-field nuclear magnetic resonance. Dynamic binding capacity of proteins on the modified supports did not significantly change with increase of the flow rate. The media showed good performance for separation three model proteins at high flow rate of 1084cm/h. This modified gigaporous microspheres had a large potential in application for rapid separation of biomolecules.
    No preview · Article · Apr 2014 · Journal of Chromatography A
  • Lan Zhao · Yongdong Liu · Yinjue Wang · Yongdong Huang · Xiunan Li · Yan Li · Guanghui Ma · Zhiguo Su
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    ABSTRACT: To study the leakage at different solution pH values, IgG Sepharose 6FF®, a commercially available immunoadsorbent, was used as a model. The leaked substance consists of three parts: (1) ligands and its fragments; (2) ligands plus matrix fragments in which ligands are chemically attached to the adsorbent matrix; and (3) matrix fragments. Buffer solution pH values had a great effect on both the kinetics and the amount of ligand leakage. Cross-linking of the adsorbent matrix could reduce both matrix leakage and antibody leakage at pH 3.0, but its effect was limited at pH 11.0 for ligand leakage. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd.
    No preview · Article · Sep 2013 · Biomedical Chromatography
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    ABSTRACT: The organometallic ruthenium(II) anticancer complexes [(η(6)-arene)Ru(en)Cl](+) (arene=p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en=ethylenediamine), exhibit in vitro and in vivo anticancer activities. In the present work, we show that they inhibit human glutathione-S-transferase π (GSTπ) with IC50 values of 59.4±1.3, 63.2±0.4 and 37.2±1.1μM, respectively. Mass spectrometry revealed that complex 1 binds to the S-donors of Cys15, Cys48 within the G-site and Cys102 at the interface of the GSTπ dimer, while complex 2 binds to Cys48 and Met92 at the dimer interface and complex 3 to Cys15, Cys48 and Met92. Moreover, the binding of complex 1 to Cys15 and Cys102, complex 2 to Cys48 and complex 3 to Cys15 induces the irreversible oxidation of the coordinated thiolates to sulfenates. Molecular modeling studies indicate that the coordination of the {(arene)Ru(en)}(2+) fragment to Cys48 blocks the hydrophilic G-site sterically, perhaps preventing substrate from proper positioning and accounting for the reduction in enzymatic activity of ruthenated GSTπ. The binding of the ruthenium arene complexes to Cys102 or Met92 disrupts the dimer interface which is an essential structural feature for the proper functioning of GSTπ, perhaps also contributing to the inhibition of GSTπ.
    Full-text · Article · Jul 2013 · Journal of inorganic biochemistry
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    Yongdong Huang · Rongyue Zhang · Juan Li · Qiang Li · Zhiguo Su · Guanghui Ma
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    ABSTRACT: Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100–500 nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5 min at a velocity up to 1400 cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.
    Full-text · Article · Jan 2013 · Protein Expression and Purification
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    Jun Wang · Yinjue Wang · Tao Hu · Xiunan Li · Yongdong Huang · Yongdong Liu · Guanghui Ma · Zhiguo Su
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    ABSTRACT: Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10kDa- and 20kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5kDa-SC-mPEG. Bioactivities of 10kDa- and 20kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.
    Full-text · Article · Jan 2012 · PROCESS BIOCHEMISTRY
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    Lan Zhao · Yongdong Liu · Yongdong Huang · Xiunan Li · Yinjue Wang · Yan Li · Guanghui Ma · Zhiguo Su
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    ABSTRACT: Hydrophobic interaction chromatography (HIC) is often an inevitable step for a satisfying purification in giant vaccine molecules production. But great mass and activity loss associated with poor purity often occur simultaneously. In this paper, high purity and high bioactivity recovery for the HIC process of hepatitis B surface antigen (rHBsAg) purification were achieved through manipulation of surface hydrophobicity of the adsorbent. Spacer arm length and ligand density were regulated, respectively, through which the interaction between the vaccine and the adsorbent was manipulated deliberately. It was found even in a narrow scope, varying spacer arm length and ligand density resulted in purification factor changing from 1 to 96.5, and rHBsAg recovery from 3 to 91%. The optimal purification performance was achieved when the spacer arm was C8 and the ligand density was 9.2 μmol/g suction-dried wet gel with an average distance of ligands of 3.6 nm. This deliberate regulation strategy represents a new approach of improving purification of giant multi-subunit proteins.
    Full-text · Article · Nov 2011 · Journal of Separation Science
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    ABSTRACT: Human plasma fraction IV is an intermediate precipitate during the production of human serum albumin using cold ethanol method. Haptoglobin locates in this fraction can be purified for various applications. A new process integration of polyethylene glycol (PEG) precipitation and ion-exchange chromatography (IEC) was developed for purification of haptoglobin, which could effectively purify the haptoglobin from 16.6% to 95%. The recovery of the new process was 58.2% in comparison to 30.3% of the conventional affinity chromatography. Furthermore, 175 mg haptoglobin production in a scaled-up process showed the method to be simple, fast, and low-cost.
    Full-text · Article · Apr 2011 · Artificial Cells Blood Substitutes and Biotechnology (formerly known as Artificial Cells Blood Substitutes and Immobilization Bi
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    Yongdong Huang · Jingxiu Bi · Lan Zhao · Guanghui Ma · Zhiguo Su
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    ABSTRACT: Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or "soft" proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183 mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant (K(a)), characteristic number of binding sites (υ) and steric factor (σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020 mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041 mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins.
    Full-text · Article · Dec 2010 · Protein Expression and Purification
  • Hang Yuan · Yan Li · Yongdong Huang · Jian Luo · Guanghui Ma · Zhiguo Su
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    ABSTRACT: As a virus-like particle, hepatitis B surface antigen (HBsAg) was the primary component of hepatitis B vaccine. HBsAg was maintained by the non-covalent interaction of proteins and lipids. The intact structure of HBsAg particle was vital to its function. However, there was no report about the effects of solvent environment on HBsAg structure. In this paper, we studied the effects of temperature, pH, ionic type and salt concentration on HBsAg structure. The results showed that HBsAg was stable at normal temperature, but began to denature above 60 degrees C. The aggregation of HBsAg at pH 3.0 and 4.0 was nearly irreversible, but partly reversible at pH 5.0. The influence of ionic type on HBsAg was generally in accordance with Hofmeister sequence, except that SO4(2-) caused more aggregation than F-. HBsAg aggregates started to be visible in 0.4 mol/L (NH4)2SO4, and the extent of aggregation increased with the salt concentration. Therefore, caution must be taken when using (NH4)2SO4 in the hydrophobic chromatography purification of HBsAg.
    No preview · Article · Dec 2010 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
  • Qian Bai · Yan Zhang · Yinjue Wang · Jian Luo · Yan Li · Yongdong Huang · Runyu Ma · Zhiguo Su
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    ABSTRACT: Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.
    No preview · Article · Nov 2010 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
  • Haixin Xing · Yongdong Huang · Yan Li · Jian Luo · Liye Zhang · Guanghui Ma · Zhiguo Su
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    ABSTRACT: Endotoxin removal is essential for the safety of biological products. To remove endotoxin efficiently, we used polymyxin B (PMB) affinity adsorbent to remove endotoxin from protein solutions by static adsorption. We studied the effects of spacer length and ligand density of the affinity adsorbent, pH, salt type and concentration, protein type and concentration, endotoxin concentration, and additive on endotoxin removal and protein recovery. Endotoxin content and protein concentration were determined by test and Lowry assay respectively. The results showed that PMB affinity adsorbent had high capacity, high adsorption speed, high removal efficiency and good reusability. In addition, ligand density, pH, salt concentration and the isoelectric point and hydrophobicity of protein all had remarkable influence on the endotoxin removal. Under the optimal conditions, the recoveries of hemoglobin, human serum albumin and lysozyme were 87.2%, 73.4% and 97.3%, respectively, and the corresponding endotoxin removal rates 99.8%, 97.9% and 99.7%, respectively. This study illustrated the effects of solution conditions on the efficiency of endotoxin removal and protein recovery, and would provide useful reference for the efficient removal of endotoxin from biological products.
    No preview · Article · Nov 2010 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
  • Tie Wu · Jingxiu Bi · Yongdong Huang · Yan Zhang · Lijing Sun · Chunbao Sun · Zhiguo Su
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    ABSTRACT: The low recovery of pertussis toxin (PT) and the low resolving efficiency of chromatography, due to the instability of PT in low salt condition, are the main challenges for its purification. We aplied 2 mol/L urea to prevent the aggregation and disassociation of PT during the purification by ion-exchange chromatography (IEC) and gel filtration chromatography (GFC). The effect of urea on the purification of PT was studied by ELISA assay and non-reduced SDS-PAGE. The activity recoveries of PT and filamentous hemagglutinin (FHA) in IEC and GFC, the resolution efficiency in GFC and the purities of PT and FHA were improved obviously by adding 2 mol/L urea in the mobile phase. The results highlight the potential application of urea in the acellular pertussis vaccine (APV) manufacture procedure.
    No preview · Article · Aug 2008 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
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    Yongdong Huang · Jingxiu Bi · Yan Zhang · Weibin Zhou · Yan Li · Lan Zhao · Zhiguo Su
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    ABSTRACT: The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.
    Full-text · Article · Jan 2008 · Protein Expression and Purification
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    Weibin Zhou · Jingxiu Bi · Lan Zhao · Yangmu Wang · Yan Li · Yongdong Huang · Guanghui Ma · Zhiguo Su
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    ABSTRACT: To improve the purification efficiency of recombinant hepatitis B surface antigen derived from Hansenula polymorpha (Hans-HBsAg), a serial of absorbents for hydrophobic interaction chromatography with the controllable ligand density and spacer arm were synthesized, then developed and further applied to purify Hans-HBsAg. The absorbent, Butyl-S QZT with the ligand density of 25 μmol/(g wet gel) and spacer arm of 3C, was screened out and its physical and chemical properties were evaluated. High rigidity and low backpressure (<0.06 MPa) were obtained at the flow rate up to 20 ml/min. Moreover, it has the stable chemical characteristics of subjecting to high concentrations of acid, alkali and detergents. This HIC absorbent was further applied to purify Hans-HBsAg with the recovery 94% and purification-fold 9 under the optimized operation condition at pH 6.5 and concentration of ammonium sulfate 7.5%. Finally, the HIC adsorbent of Butyl-S QZT was applied in the integrated three-step chromatographic purification process to purify Hans-HBsAg. About 140 mg of purified Hans-HBsAg was obtained from 1 l cell disruption supernatant at the total recovery of 27% and the purification-fold of 151.8. Based on the assay of SDS-PAGE and SEC-HPLC, the purity of the purified HBsAg was over 99% to meet the requirement for the further inoculation use.
    Full-text · Article · May 2007 · PROCESS BIOCHEMISTRY