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Zhiguo Su

Publications and source records attributed to Zhiguo Su.

29 records · Page 2Linked to original sources
PubMed2004

Purification of hemoglobin by ion exchange chromatography in flow-through mode with PEG as an escort.

Development of hemoglobin-based blood substitutes requires production of highly purified hemoglobin. Process of hemoglobin purification by ion exchange chromatography in flow-through mode was researched and optimized. Three kinds of media including, QMA Spherosil LS (Biosepra, France) and Q Sepharose Big Beads (Amersham Bioscience, Sweden), and an anion exchange membrane column, Mustang Q (PALL, USA) were investigated and compared. Adding polyethylene glycol (PEG) as an escort in ion exchange chromatography improved the purity and recovery, and the recovery in the chromatography was increased from 75 to 95%. The mechanism of PEG effects on chromatography was discussed. The optimal chromatography step, in combination with hypotonic dilution hemolyzing and membrane separation, formed an integrated hemoglobin purification process. The total recovery in the process was 87.6%. The activity of hemoglobin was well preserved: P50 23.2 mmHg, and Hill coefficient 2.31. The product appeared as a single band in SDS-PAGE, and GF-HPLC showed only one peak. The purity of the prepared hemoglobin was more than 99.9%. The optimized process is time saving and suitable for large-scale preparation of hemoglobin to provide materials for further preparation of blood substitutes.

Animals↗
PubMed2003

Inhibition of aggregation by media selection, sample loading and elution in size exclusion chromatographic refolding of denatured bovine carbonic anhydrase B.

The effects of gel media, sample application and elution flowrate on the activity recovery and aggregation in refolding of bovine carbonic anhydrase B (CAB) by size exclusion chromatography (SEC) were investigated. Variation in aggregation was demonstrated visibly by the comparison of refolding profiles under different operating conditions. Some principles with regard to practical application were proposed. Meanwhile, the analysis of relationship between peak resolution and activity recovery provided evidences for the mechanism of size exclusion chromatography protein refolding.

Animals↗
PubMed2003

Separation and identification of different refolding components.

A dual-gradient ion-exchange chromatography (IEC) refolding process has been successfully applied to renature lysozyme at high concentration. The different refolding components were separated by size-exclusion chromatography with low concentrations of urea and NaCl added into elution buffer. The hydrodynamic characteristics during separation and the hydrophobic properties of these components were also tested.

Chromatography, Ion Exchange↗
PubMed2003

A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin conjugate.

A solid phase adsorption method was proposed to prepare well-defined bovine serum albumin-bovine hemoglobin (Hb) conjugate. After adsorption by the solid phase, Q Sepharose Fast Flow media, bovine serum albumin (BSA) molecules were allowed to react with glutaraldehyde. The spacing out of BSA molecules on the solid phase was assumed to limit polymerization of BSA molecules, except some molecules bound closely on the solid phase resulting in minor dimer formation. Following the elution procedure, the activated monomeric BSA was separated from the dimers by gel filtration chromatography on Superdex 200 and then reacted with bovine Hb at 4 degrees C and pH 9.5. The 1:1 (BSA:Hb) conjugate was obtained with the yield of 64%. The P(50) values of the conjugates, prepared under anaerobic and aerobic conditions, were 19.1 and 14.2 mmHg, respectively. The dependence of the P(50) on chloride ions for the conjugate was slightly diminished, presumably due to covalent attachment of BSA to bovine Hb.

Adsorption↗
PubMed2003

Stability of tumor necrosis factor-alpha during ion exchange chromatography.

Denaturation of target protein by various separation and purification steps contributes significant part to the total product loss in bioseparation. The conformational change and accompanying loss of activity of tumor necrosis factor-alpha during ion exchange chromatography was reversible and was decreased by adding polyethylene glycol 200 at 2 to 5% (v/v) to the eluting solution.

Chromatography, Ion Exchange↗
PubMed2003

Refolding of a recombinant full-length non-structural (NS3) protein from hepatitis C virus by chromatographic procedures.

The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) was expressed as inactive aggregates in Escherichia coli. The protein was refolded by chromatographic techniques of which ion exchange chromatography was best for crude samples and gel filtration best for partially purified samples. Immobilized metal ion affinity chromatography showed intermediate performance. Gradient procedures enhanced the recovery of active NS3 protein.

Chromatography↗
PubMed2003

Preparation and characterization of dimeric bovine hemoglobin tetramers.

Dimeric bovine hemoglobin (Hb) tetramers were prepared by a one-step solid phase adsorption method. Briefly, Hb was absorbed by the solid phase, Q Sepharose Fast Flow media, followed by reaction with the glutaraldehyde and elution procedure. Then, dimeric bovine Hb tetramers were formed and purified from Hb tetramers by anion-exchange chromatography based on Protein-Pak DEAE 8HR. The dimeric Hb tetramer showed a P50 value of 15.9 mm Hg, oxygen transporting efficiency of 14.2%, and Hill coefficient of 1.72. The number of Bohr protons released for dimeric Hb tetramers was 0.59 H/tetramer, which was 39% of that of native bovine Hb. The number of chloride ions released on oxygenation was 0.60/tetramer for dimeric Hb tetramers, which was 46% of that of native bovine Hb.

Animals↗
PubMed2002

Dual gradient ion-exchange chromatography improved refolding yield of lysozyme.

Protein refolding at high concentrations always leads to aggregation, which limits commercial application. An ion-exchange chromatography process with gradient changes in urea concentration and pH was developed to refold denatured lysozyme at high concentration. After adsorption of the denatured protein onto an ion-exchange medium, elution was carried out in combination with a gentle decrease in urea concentration and elevation of pH. Protein would gradually refold along the column with high activity yield. Denatured and reduced lysozyme at 40 mg/ml was loaded into a column filled with SP Sepharose Fast Flow, resulting in 95% activity recovery and 98% mass yield within a short period of time.

Chromatography, Ion Exchange↗
PubMed2002

Preparation of well-defined bovine polyhemoglobin based on dimethyl adipimidate and glutaraldebyde cross-linkage.

A well-defined bovine polyhemoglobin was prepared by dimethyl adipimidate (DMA) and glutaraldehyde double cross-linkage method. DMA was used to block some amino groups of hemoglobin, followed by further polymerization with glutaraldehyde. The amino modification degree of hemoglobin was 32% when DMA reacted with hemoglobin at the molar ratio of 200. The bovine polyhemoglobin with narrow molecular weight distribution (mainly 128 kDa) was obtained when glutaraldehyde reacted with DMA-modified hemoglobin. The P(50) and the Hill coefficient for DMA-modified hemoglobin were 19.4 mm Hg and 2.28, respectively, while those for the bovine polyhemoglobin were 15.1 mm Hg and 1.70, respectively. The number of Bohr protons released for DMA-modified hemoglobin and the polyhemoglobin was 0.86 and 0.56 H/tetramer, respectively.

Animals↗
PubMed2002

Preparation and characterization of PEGylated adducts of recombinant human tumor necrosis factor-alpha from Escherichia coli.

This study was conducted to get an insight into monomethoxypolyethylene glycol (MPEG) modified recombinant human TNF-alpha (rhTNF-alpha) derived from gene cloning and expression. The purification and modification processes were integrated together to make the scale-up production of PEG-modified rhTNF-alpha practical. Capillary electrophoresis was demonstrated to be a highly efficient tool in the biochemical characterization of the PEGylated products compared to slab electrophoresis. The cytotoxicity of MPEG-modified rhTNF-alpha was studied in vitro towards L929 cell line and found to decrease gradually along with the increase in the reaction ratio between the activated MPEG and the native rhTNF-alpha. The MPEG-modified rhTNF-alpha was more resistant to proteinase degradation in vitro than the native. When MPEG chains were released partially from the MPEG-modified rhTNF-alpha by alkaline pretreatment, the cytotoxicity of the MPEG-modified rhTNF-alpha was enhanced, which was in contrast to the native. These results would be helpful to explain the disagreement between the high bioavailability of MPEG-modified TNF-alpha in vivo and its decreased cytotoxicity in vitro.

Animals↗