生物分离工程双语版3
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(5) activity loss A final fractionation B previous bioseparation steps
Exmaple 3.1 carboxylesterase purification
❖ (1) Material ：Bacillus stearothermophilus噬热脂杆菌
❖ B Two bands of higher mobility (after precipitation and anion
❖
exchange）
❖ C just single band of high mobility (further gel filtration)
❖ D gel filtration and SDS-PAGE indicate esterase is
II Chromatography procedures
❖A gel filtration chromatography ❖B Hydroxylapatite chromatography ❖C Hydrophobic interaction ❖ chromatography ❖D Affinity chromatography ❖E Ion-Exchange Chromatography
Difference between crude and purified esterase forms
❖ (1) Thiol-containing compounds in crude ❖ material lead to cysteine-containing protein ❖ denaturation ❖ (2) Proteolysis activity in crude material ❖ (3) Coprecipitation with less stable compounds ❖ in crude material ❖ (4) Conformational changes in processing ❖ steps
Chapter 3 1st
High resolution fractionation processes
I Introduction
❖ (1) appropriate sequence ❖ (2) utilization (therapeutic, diagnostic) ❖ (3) operation scale ❖ (4) role of final fractionation ❖ A impurities ❖ B salts ❖ C pathogens ❖ D endotoxins ❖ E contaminating proteins ❖ (5) low volume and expensive procedures ❖ (6) special attention ❖ A amount of product denaturation ❖ B quality
❖
monomeric protein with MW 40000
Exmaple 3.1 carboxylesterase purification
❖ (4) Reason ❖ A active monomer associating multiple forms ❖ B multiple form have different chromatography and ❖ electrophoretic properties ❖ C reaction equilibrium affected by pH, salt ❖ concentration, enzyme dilution ❖ D monomers or multimeric enzyme forms affect ❖ activity and stability
A gel filtration chromatography
❖ (1) mechanism ❖ A molecular size ❖ B mobile solvent phase ❖ C stationary phase ❖ (2) Rigidity of beads in large scale operation ❖ (3) traditional gel filtration chromatography media ❖ A Sephadex ❖ B Superose ❖ C Sephacryl ❖ D Trisacryl ❖ E Biogel P ❖ F Cellulofine ❖ G Fractogel ❖ (4) low product volume suitable for gel filtration
Results and discussion about table 3.1
❖ (1) 48% recovery is high yield (not so high ❖ purification factor) ❖ (2) State change of enzyme aggregation lead ❖ to activity loss ❖ (3) Analyze cause permit minimize activity loss
❖ (2) Step utilized
❖ A centrifugation
❖ B DEAE-Sephacel ion-exchange chromatography
❖ C gel filtration chromatography
❖ (3) Result analysis
❖ A single low-mobility band (PAGE)
wk.baidu.com
Purification of antigenized immunoglobulins
with monomethoxypolyethylene glycol
(1) polyethylene glycol (PEG) usage ❖ A nontoxic ❖ B nonimmunogenic ❖ C internal （内用）use in humans ❖ D PEGylated proteins preserve biological activity (2) various degree of derivatization occur
Exmaple 3.1 carboxylesterase purification
❖ (1) Material ：Bacillus stearothermophilus噬热脂杆菌
❖ B Two bands of higher mobility (after precipitation and anion
❖
exchange）
❖ C just single band of high mobility (further gel filtration)
❖ D gel filtration and SDS-PAGE indicate esterase is
II Chromatography procedures
❖A gel filtration chromatography ❖B Hydroxylapatite chromatography ❖C Hydrophobic interaction ❖ chromatography ❖D Affinity chromatography ❖E Ion-Exchange Chromatography
Difference between crude and purified esterase forms
❖ (1) Thiol-containing compounds in crude ❖ material lead to cysteine-containing protein ❖ denaturation ❖ (2) Proteolysis activity in crude material ❖ (3) Coprecipitation with less stable compounds ❖ in crude material ❖ (4) Conformational changes in processing ❖ steps
Chapter 3 1st
High resolution fractionation processes
I Introduction
❖ (1) appropriate sequence ❖ (2) utilization (therapeutic, diagnostic) ❖ (3) operation scale ❖ (4) role of final fractionation ❖ A impurities ❖ B salts ❖ C pathogens ❖ D endotoxins ❖ E contaminating proteins ❖ (5) low volume and expensive procedures ❖ (6) special attention ❖ A amount of product denaturation ❖ B quality
❖
monomeric protein with MW 40000
Exmaple 3.1 carboxylesterase purification
❖ (4) Reason ❖ A active monomer associating multiple forms ❖ B multiple form have different chromatography and ❖ electrophoretic properties ❖ C reaction equilibrium affected by pH, salt ❖ concentration, enzyme dilution ❖ D monomers or multimeric enzyme forms affect ❖ activity and stability
A gel filtration chromatography
❖ (1) mechanism ❖ A molecular size ❖ B mobile solvent phase ❖ C stationary phase ❖ (2) Rigidity of beads in large scale operation ❖ (3) traditional gel filtration chromatography media ❖ A Sephadex ❖ B Superose ❖ C Sephacryl ❖ D Trisacryl ❖ E Biogel P ❖ F Cellulofine ❖ G Fractogel ❖ (4) low product volume suitable for gel filtration
Results and discussion about table 3.1
❖ (1) 48% recovery is high yield (not so high ❖ purification factor) ❖ (2) State change of enzyme aggregation lead ❖ to activity loss ❖ (3) Analyze cause permit minimize activity loss
❖ (2) Step utilized
❖ A centrifugation
❖ B DEAE-Sephacel ion-exchange chromatography
❖ C gel filtration chromatography
❖ (3) Result analysis
❖ A single low-mobility band (PAGE)
wk.baidu.com
Purification of antigenized immunoglobulins
with monomethoxypolyethylene glycol
(1) polyethylene glycol (PEG) usage ❖ A nontoxic ❖ B nonimmunogenic ❖ C internal （内用）use in humans ❖ D PEGylated proteins preserve biological activity (2) various degree of derivatization occur