Oligomeric or polymerized methylene protein separation and purification process
A protein separation, separation and purification technology, applied in the field of protein preparation, can solve the problems of dissociation that have not been reported in any reports, and achieve the effects of protecting protein activity and separation and purification effects, reducing losses, and protecting integrity
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Embodiment 1
[0029] Example 1 Extraction of alcohol dehydrogenase (tetrameric protein) from yeast
[0030] Yeast: Commercially available dry baker's yeast (Gist-Brocades Yeast Co., Ltd., Netherlands) was dissolved in distilled water at a yeast concentration of 10% weight / volume (W / V) (dry weight), and 8 liters of yeast suspension was prepared. Stir for one hour at 4 °C to activate the yeast.
[0031] Adding protectant: After one hour, the cell suspension was divided into two parts (4 liters each), and 1 liter of glycerol and 20% (W / V) sucrose was added to one part of the activated yeast suspension under stirring. The solution was mixed to make the suspension volume 5 liters; another cell suspension without any protective agent was supplemented with distilled water to 5 liters as a reference comparison.
[0032] Broken cells: Use a high-pressure homogenate pump produced by APV Manton Gaulin Company in the United States to add five liters of raw material solution into the feeding tank and s...
Embodiment 2
[0035] Example 2 Extraction of human tumor necrosis factor (trimer) from genetically engineered Escherichia coli
[0036] Escherichia coli (E.coli cells) culture: recombinant human tumor necrosis factor (rhTNF-α) Escherichia coli JM103, the medium is 1.5% weight / volume (W / V) tryptone, 1.3% weight / volume (W / V) Yeast extract powder, 0.25 weight / volume (W / V) NaCl, 0.2% weight / volume (W / V) glucose, pH 7.5 The final concentration of tetracycline was 12.5 μg / l. The fermentation of the recombinant bacteria was carried out at 37° C. with a 6-liter fermenter (product of New Brunswick Scientific, Co., USA, model BIOFLO II), and the fermentation period was 12 hours. After fermentation, the E.coli cells were collected by centrifugation at 4,500 g for 20 min. The cells were washed with a buffer solution of 10 mmol Tris CH1, 1 mmol EDTA, pH 8.0, and then suspended with the same buffer solution to form a cell suspension with a concentration of 20 wt%.
[0037] Adding protective agent: divi...
Embodiment 3
[0043] Example 3 Purification of genetically engineered human tumor necrosis factor by adsorption
[0044] Adsorption: The anion exchange medium DEAE-Sepharoes C1-6B was loaded into an adsorption column (16×55mm), and fully equilibrated with 0.01M Tris-HCl, pH 8.0 (with or without polyethylene glycol PEG). Add polyethylene glycol PEG200, 600 or 4000 at a concentration of 1 wt% to the cell disruption supernatant, balance solution, and eluate without the presence of polyethylene glycol PEG, respectively. Feed, wash with equilibration buffer to remove unadsorbed protein to the baseline, use 75mM NaCl, 10mM Tris-HCl for segmental elution, collect each elution peak and measure protein concentration and RhTNF-α activity.
[0045] Activity analysis: TNF activity analysis refers to the method of Aggarwal et al [Aggarwal, B.B., Kohr, w.j., Hass, P.E., Moffar, B., Spence, S.A., Henzel, W.J., Bringman, T.S., Nedwin, G.E., Goeddel, D.V.and Harkins, R.N.J. Biol. Chem. 260, 2345-2354, 1985...
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