Biomedical subjects

Xindu Geng

Publications and source records attributed to Xindu Geng.

11 recordsLinked to original sources

PubMed2006

Protein folding liquid chromatography and its recent developments.

The ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. However, proteins produced by Escherichia coli must be refolded to their native state. Protein folding liquid chromatography (PFLC) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. In this paper, the new method, PFLC is introduced and its recent development is reviewed. In addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of PFLC. In addition, the role of this method in the future is examined.

Chromatography, Liquid↗

PubMed2006

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers.

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.

Adsorption↗

PubMed2006

[Preparation of a concanavalin A immobilized affinity column and its application in the structural analysis of ribonuclease B].

The research on glycoproteomes represents an interesting field in the functional proteomics research. Affinity chromatography and mass spectrometry are powerful techniques that are used for gaining valuable information on glycoproteomes because glycoproteins and their unusual forms resulting from protein glycosylation can be important indicators of several diseases. In this study, the concanavalin A (Con A) immobilized silica packing was prepared and used for the separation of glycoprotein and glycopeptides. A very low, non-specific adsorption on the Con A affinity column was demonstrated by mass recovery of bovine serum albumin at more than 98.5%. The effect of concentration of methyl-alpha-D-mannopyranoside (alpha-Me-D-Man) in the mobile phase and the effect of flow rate on the retention behavior of ribonuclease B (RNase B) were also investigated. The standard glycoprotein RNase B was separated under optimized conditions using 0.2 mol/L alpha-Me-D-Man in the mobile phase at a flow rate of 0.5 mL/min. Meanwhile, the oligosaccharides and glycopeptides were enriched using a Con A column after digestion of the purified RNase B with peptide-N-glycosidase F (PNGase F) and trypsin. The structure of N-linked glycan and the rate and the site of glycosylation of RNase B were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Glycoproteins and glycopeptides in human serum and digest solution could be separated by this method. The results showed that this method is rapid and sensitive for the purification and characterization of glycoproteins and glycopeptides.

Animals↗

PubMed2006

[Determination of puerarin in Kangshen granule by reversed-phase high performance liquid chromatography].

Kangshen granule, containing 12 kinds of traditional Chinese medicine, has complex composition. A new method for the determination of puerarin in Kangshen granule was developed using reversed-phase high performance liquid chromatography coupled with matrix assisted laser desorption ionization time of flight mass spectrometry and ultraviolet detection (RP-HPLC/MALDI-TOF MS/UV). The sample was separated by RP-HPLC with a non-linear gradient elution, and then was qualitatively detected by mass spectrometry to identify the puerarin peak. The chromatographic process was performed on a YMC-ODS column under the optimized conditions with a mobile phase consisting of methanol, water and 0.1% TFA, and detected at 250 nm. The linear range of puerarin was from 0. 132 to 1.32 microg (r = 0.9997). The recovery was higher than 103% and the relative standard deviation was lower than 2.0% (n=6). The puerarin content in Kangshen granule sample was determined and the puerarin content was 3.09 mg/g. The method is simple and feasible for the determination of puerarin with a good repeatability. The method can be used for quality control of Kangshen granule.

Chromatography, High Pressure Liquid↗

PubMed2004

Refolding and purification of interferon-gamma in industry by hydrophobic interaction chromatography.

A new technology for renaturation with simultaneous purification of the recombinant human interferon-gamma (rhIFN-gamma) in downstream of biotechnology is presented. The strategies to develop the new technology in industry scale were suggested. Based on chemical equilibrium and molecular interactions, the principle of rhIFN-gamma refolding by HPHIC was described. The kind of stationary and mobile phases were evaluated and found the former to contribute to the rhIFN-gamma refolding more than the latter. The extract containing the rhIFN-gamma in gram scale in 7.0 mol L(-1) guanidine hydrochloride solution of 700 mL was directly pumped into a unit of simultaneous renaturation and purification of proteins (USRPP, 10 x 300 mm i.d.) packed by small particle packings of hydrophobic interaction chromatography and a satisfactory recovery of bioactivity and mass of the rhIFN-gamma was obtained. With flow rate 100 mL min(-1) and a gradient elution by only one step in 4h, the purity and specific bioactivity approach to 95% and 8.7 x 10(7) IU(-1) mg, respectively. To evaluate the goodness of the presented new technology in this study, a usual method with the renaturation by dilution method firstly and then purification with a series of LC in literature was employed to compare with each other. The obtained result in terms of purity, recoveries of mass and bioactivity, cost time as well as expenses, the former is much better than the latter. Comparing the total bioactivity of rhIFN-gamma in the extract before to that after the renaturation by the USRPP, the total bioactivity of rhIFN-gamma increased 62-fold.

Biotechnology↗

PubMed2004

Purification of recombinant bovine normal prion protein PrP(104-242) by HPHIC.

Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104-242, PrP(104-242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104-242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104-242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104-242) by HPHIC. This procedure yield PrP(104-242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min.

Animals↗

PubMed2004

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application.

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.

Biopolymers↗

PubMed2004

Design and resolution of a simple-type column for renaturation with simultaneous purification of proteins and its application to lysozyme refolding.

A new kind of column called simple-type chromatographic column using the packings of large particles, which resembles columns used in traditional low pressure liquid chromatography in both shape and operation but has resolution of biopolymers similar to high performance liquid chromatography, was designed and investigated. In addition, so long as a suitable stationary phase, such as hydrophobic interaction chromatographic stationary phase, is packed, the column can be employed for the purification with simultaneous renaturation of proteins. The structure, operation and characteristics of the designed column including pressure drop, column life, and resolution were investigated. Lysozyme was selected as the model protein for investigating the renaturation. The recoveries of mass and bioactivity were found to be (96.6 +/- 1.3)% and (101.1 +/- 6.0)% respectively when the original concentration of lysozyme was 50.0 g/L. The simple-type column is very cheap and easy to use, and can be applied to the purification of proteins under the condition of low pressure. Thus, it has great potential applications for the purification and/or renaturation of proteins in large scale, down-stream processing of therapeutic proteins in biotechnology.

Chromatography, High Pressure Liquid↗

PubMed2004

A fast method for preparation of cytochrome-C of high purity from pig heart by hydrophobic interaction chromatography.

A fast method for the preparation of cytochrome C (Cyt-C) of high purity from pig heart is presented. A simple-type column of hydrophobic interaction chromatography packed with big particles was employed for the fast purification in laboratory scale. The purity of the purified Cyt-C was verified by reversed-phase liquid chromatography, UV spectroscopy, and iron content in it. Compared with the common method, the new method has advantages of higher speed, simpler purification processes, higher mass recoveries and higher purity.

Animals↗

PubMed2004

[Fast purification of human serum albumin with chromatographic pie in analytical scale].

The purification of human serum albumin (HSA) by using analytical chromatographic pie was developed. Six standard proteins were separated on an analytical chromatographic pie. The results from the resolution of the standard proteins under various flow rates indicate that the chromatographic pie in analytical scale packed with small particles can be utilized under fast flow conditions. The type of mobile phase, concentration of (NH4)2SO4 and pH value for HSA purification were optimized. Crude HSA solution was purified in 10 min in analytical scale with the purity of HSA more than 85% and recovery of 65%. The results show that analytical chromatographic pie is suitable for fast separation and purification of biopolymers.

Chromatography, High Pressure Liquid↗

PubMed2003

Preparation of weak cation exchange packings based on monodisperse poly(glycidyl methacrylate- co-ethylene dimethacrylate) beads and their chromatographic properties.

The monodisperse, macroporous poly(glycidyl methacrylate- co-ethylene dimethacrylate) beads were synthesized by a single-step swelling and polymerization method. Based on this media, a weak cation exchange (WCX) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery, and good resolution for proteins. The measured bioactivity recovery for lysozyme was 98+/-5%. The dynamic protein loading capacity of the WCX packings was 17.3 mg g(-1). The experimental results show that the synthesized WCX resin has very weak hydrophobicity.

Biopolymers↗