CN101367864B - Separation purification process for heterogenous homogeneous serum albumin - Google Patents
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Abstract
The present invention relates to a method for separating and purifying heterogenous and homogeneous whey proteins. The method includes the following steps: genetically modified cow's milk containing the heterogenous and homogeneous whey proteins is chosen as material; whey is obtained after lipid and casein are removed by centrifugation; the obtained whey is preprocessed, removing macromolecular impurities such as immune globulin; the whey or the preprocessed whey undergoes hydrophobic interaction chromatography separation; the heterogenous and homogeneous whey proteins in the whey are interacted with hydrophobic chromatography media; because the spacial conformations and hydrophobicity of the heterogenous and homogeneous whey proteins are different, the heterogenous and homogeneous wheyproteins can be separated in hydrophobic chromatography; and further, the pure products of the heterogenous and homogeneous whey proteins with high purity can be obtained by gel permeation chromatography. The method has the advantages of high recovery rate and stable production technique and is suitable for mass production.
Description
Technical field
The invention belongs to proteic separation purification method, particularly relate to a kind of separation purification method of heterogenous homogeneous serum albumin.
Background technology
Many whey-proteins in the Mammals emulsion contain higher nutritive value and pharmaceutical use, as various indispensable amino acids, antibacterial and inducing apoptosis of tumour cell are provided for body.There is anaphylactogen in whey-protein in Mammals emulsion such as the cow's milk, can cause part infant and grownup that the anaphylaxis of IgE or non-IgE mediation takes place.Utilize transgenic technology express transgenic people source whey-protein in the mammal galactophore reactor, can improve the nutritive value of emulsion, the transgenosis emulsion can be used as the foodstuff additive of high nutritive value, high economic value added as the people source whey-protein of feed purification.
In the transgene mammal emulsion except that the people source whey-protein of transgene expression, the whey-protein that also has the Mammals oneself expression, the heterogenous homogeneous serum albumin structural similitude, physico-chemical property is approaching, be difficult to separate, Wang Jianwu (identify and analyze China Agricultural University's Ph D dissertation by the recombination human alpha-whey albumin transgenic cattle, 2007) report separates Niu Yuan and people source alpha-whey albumin with the method for anionresin isocratic elution, but is difficult to effectively amplify.
Summary of the invention
The separation purification method that the purpose of this invention is to provide a kind of heterogenous homogeneous serum albumin.
The technical solution used in the present invention step is as follows:
1) choosing the transgene mammal emulsion that contains a kind of heterogenous homogeneous serum albumin at least is raw material, collects supernatant liquor behind centrifugal removal lipid and the casein and obtains whey;
2) the gained whey is removed macromole impurity such as immunoglobulin (Ig), remaining casein through pre-treatment;
3) with the combination chromatography technology of whey or pretreated whey process hydrophobic interaction chromatography and gel permeation chromatography coupling or through single step hydrophobic interaction chromatography technology, separate obtaining heterogenous homogeneous serum albumin.
Transgene mammal emulsion of the present invention is preferably cow's milk, and described heterogenous homogeneous serum albumin is preferably ox source alpha-whey albumin, Lactotransferrin, the N,O-Diacetylmuramidase of transgenic human source alpha-whey albumin, Lactotransferrin, N,O-Diacetylmuramidase and ox oneself expression; Below only be illustrated, but protection of the present invention is not limited to therewith with a kind of proteic separation; Total protein concentration is 30-38g/L in the cow's milk, heterogenous homogeneous serum albumin total concn 1.0-4.0g/L, and the optimization protein total concn is 1.5-3.5g/L.
Whey pre-treatment step of the present invention can adopt the method for ammonium sulfate precipitation or ultrafiltration.When adopting ammonium sulfate precipitation, need at room temperature whey to be regulated pH to 5.0-10.0, preferred pH value is 6.0-8.0, and make that by adding the ammonium sulfate powder ammonium sulfate saturation ratio is 20%-60% in the whey, count 0.86-2.95M with mole number, preferred ammonium sulfate saturation ratio is 30%-60%, counts 1.33-2.37M with mole number, vibrated centrifugal collection supernatant liquor 2 hours.When adopting ultrafiltration, need whey is regulated the pH value to 5.0-10.0, preferred pH value is 6.0-8.0, preferably adopts the ultra-filtration membrane ultrafiltration of molecular weight cut-off 30-200KD, collects filtrate.
In the hydrophobic interaction chromatography of the present invention, it is matrix that the hydrophobic interaction chromatography medium adopts the agarose material, and described aglucon can be butyl, phenyl, octyl group, is preferably butyl.
In the hydrophobic interaction chromatography of the present invention, adoptable hydrophobic salt is ammonium sulfate, sodium sulfate, sodium-chlor etc., preferably adopts ammonium sulfate;
In the hydrophobic interaction chromatography of the present invention, buffer solution system can adopt phosphate buffered saline buffer, tris-HCI buffer, Sodium phosphate dibasic-citrate buffer solution, is preferably phosphate buffered saline buffer; Low salt buffer is preferably 20-100mM, and the phosphate buffered saline buffer of pH6.0-8.0, damping fluid do not have hydrophobic salt or hydrophobic salt concentration below 0.5M; High-salt buffer is preferably 20-100mM, the phosphate buffered saline buffer of pH6.0-8.0, and hydrophobic salt concentration is 0.5-2.0M, preferred concentration is 0.6-1.2M.
In the hydrophobic interaction chromatography of the present invention, the whey solution that pre-treatment obtains is regulated pH to 5.0-10.0, preferred pH value is 6.0-8.0; And whey solution is regulated hydrophobic salt concentration is 0.5-2.0M, and preferred concentration is 0.6-1.5M, and hydrophobic salt concentration can adopt low salt buffer or hydrophobic salt powder to regulate.
In the hydrophobic interaction chromatography of the present invention, those skilled in the art can suitable hydrophobic chromatoghaphy medium, chromatography column and sample introduction speed will be through pretreated whey solution sample introduction to using in the good hydrophobic chromatography post of high-salt buffer balance according to the type selecting of heterogenous homogeneous serum albumin, the chromatography process uses 280nm uv-absorbing wavelength to carry out Protein Detection, the whey solution volume of sample introduction is a 0.5-5.0 times of chromatography column volume, is preferably 1.0-3.0 times of chromatography column volume; A kind of in the whey in heterogenous homogeneous serum albumin allosteric under suitable high salt concentration combines with the aglucon of hydrophobic medium and is retained in the post, low hydrophobic another kind of heterogenous homogeneous serum albumin and other impurity albumen then can't keep, after all sample feeding finishes, remove the albumen that do not keep in the hydrophobic interaction chromatography post with high-salt buffer flushing, get back to baseline up to the uv-absorbing wavelength; Use the low salt buffer wash-out again, type of elution can be one-step elution, stepwise elution, linear wash-out, collect elutriant to obtain a kind of of heterogenous homogeneous serum albumin, those skilled in the art can regulate preliminary purification product or the pure product of processing parameter to obtain different heterogenous homogeneous serum albumins; Because the hydrophobicity of heterogenous homogeneous serum albumin is strong and weak different, heterogenous homogeneous serum albumin is separated by this step.
In the gel permeation chromatography of the present invention, the preliminary purification product of different heterogenous homogeneous serum albumins is removed residual impurity to obtain pure product through gel permeation chromatography again, the gel permeation chromatography medium is the agarose medium, is preferably Superdex 75.
The present invention is with respect to prior art, owing to adopted the hydrophobic chromatography technology, can the processing condition of concrete hydrophobic chromatography be can regulate according to the differential liberation of conformation between the heterogenous homogeneous serum albumin and hydrophobic property, different heterogenous homogeneous serum albumin preliminary purification products or pure product obtained; Adopt gel permeation chromatography that the preliminary purification product has been carried out further making with extra care purifying, obtained the pure product of highly purified heterogenous homogeneous serum albumin; Adopt direct method, simplified and removed caseic step, improved the rate of recovery of the whey-protein in the cow's milk preprocessing process; Employing is the combination technological method of core with the hydrophobic chromatography, and stable processing technique is suitable for carrying out the large-scale production of transgenosis heterogenous homogeneous serum albumin, product purity height, technology rate of recovery height.
Description of drawings
Fig. 1 is the separation purifying technique schema
A is the process flow sheet that whey is carried out hydrophobic chromatography after treatment
B is the process flow sheet that whey directly makes up chromatography
Fig. 2 is Butyl Sepharose 4FF hydrophobic chromatography separation and purification people source a-LA.
Fig. 3 is Superdex 75 gel-filtration separation and purification people source a-LA.
Fig. 4 is a SDS-PAGE identifier source a-LA collection of illustrative plates.
1 is the pretreated whey of process, and 2 for hydrophobic chromatography penetrates peak p0, and 3 for the hydrophobic chromatography elution peak is the pure product of people source a-whey albumin, and 4 is that gel-filtration peak p3 is the pure product of ox source a-whey albumin
Fig. 5 is an anti-phase identifier source a-LA liquid phase collection of illustrative plates, and chromatographic column is C4protein, detects wavelength 280nm.
Fig. 6 is a mass spectrum identifier source a-LA collection of illustrative plates.
Get 100mL transgenic human source a-whey albumin cow's milk, the cow's milk total protein concentration is 33g/L, people source a-whey albumin concentration is 1.4g/L, ox source a-whey albumin concentration is 1.0g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.5g/L, people source a-whey albumin concentration is 1.7g/L, and ox source a-whey albumin concentration is 1.2g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 8.0, slowly adding the ammonium sulfate powder is 45% to whey ammonium sulfate saturation ratio, with the mole numeration is 2.09M, after at room temperature vibrating 2 hours, deposit sample is used under 4 degrees centigrade in the Sigma 3k30 whizzer, and to leave mental and physical efforts under order be centrifugal 30 minutes of 10000g and collect supernatant liquor 70mL, supernatant liquor total protein concentration 4.5g/L, people source a-whey albumin concentration is 1.8g/L, and ox source a-whey albumin concentration is 1.2g/L.
Use 20mM, the pH7.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 0.8M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 7.0, sample introduction is to using 20mM, pH7.0 contains in the good Butyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 0.8M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GEAKTAExplorer100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH7.0 to contain 0.8M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 150mL in the collection, total protein concentration is 1.4g/L, and people source a-whey albumin concentration is 0.01g/L, and ox source a-whey albumin concentration is 0.5g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH7.0 is also collected elution peak 105mL, and total protein concentration is 1.0g/L, and people source a-whey albumin concentration is 0.95g/L, purity 95%, and the technology rate of recovery 71%, ox source a-whey albumin concentration is 0.02g/L.
Get and penetrate peak 30mL sample introduction to using 20mM, pH7.0 contains in the good Superdex 75 gel permeation chromatography posts of the phosphate buffered saline buffer pre-equilibration of 0.1M sodium sulfate, 3.5 centimetres of column internal diameters, 60 centimetres of post bed heights, tomography devices GE AKTA Explorer 100, ultraviolet 280nm wavelength absorption detects, the phosphate buffered saline buffer that continues after flow velocity 2mL/min, last sample finish to use 20mM, pH7.0 to contain 0.1M sodium sulfate washes, the protein peak of collecting the 330mL place is total to 80mL, total protein concentration is 0.18g/L, and people source a-whey albumin concentration is 0.005g/L, and ox source a-whey albumin concentration is 0.175g/L, purity 97%, the technology rate of recovery 70%.
Get 100mL transgenic human source a-whey albumin cow's milk, the cow's milk total protein concentration is 33g/L, people source a-whey albumin concentration is 1.4g/L, ox source a-whey albumin concentration is 1.0g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.5g/L, people source a-whey albumin concentration is 1.7g/L, and ox source a-whey albumin concentration is 1.2g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 7.0, slowly adding the ammonium sulfate powder is 40% to whey ammonium sulfate saturation ratio, with the mole numeration is 1.84M, after at room temperature vibrating 2 hours, deposit sample is used under 4 degrees centigrade in the Sigma 3k30 whizzer, and to leave mental and physical efforts under order be centrifugal 30 minutes of 10000g and collect supernatant liquor 70mL, supernatant liquor total protein concentration 5.4g/L, people source a-whey albumin concentration is 1.85g/L, and ox source a-whey albumin concentration is 1.3g/L.
Use 20mM, the pH6.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 0.7M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 6.0, sample introduction is to using 20mM, pH6.0 contains in the good Octyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 0.7M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GEAKTA Explorer 100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH6.0 to contain 0.7M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 160mL in the collection, total protein concentration is 1.2g/L, and people source a-whey albumin concentration is 0.01g/L, and ox source a-whey albumin concentration is 0.5g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH6.0 is also collected elution peak 108mL, and total protein concentration is 1.7g/L, and people source a-whey albumin concentration is 1.0g/L, purity 58%, and the technology rate of recovery 77%, ox source a-whey albumin concentration is 0.1g/L.
Get 100mL transgenic human source a-whey albumin cow's milk, the cow's milk total protein concentration is 33g/L, people source a-whey albumin concentration is 1.4g/L, ox source a-whey albumin concentration is 1.0g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.5g/L, people source a-whey albumin concentration is 1.7g/L, and ox source a-whey albumin concentration is 1.2g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 7.0, use the DDB-300 of the letter Instr Ltd. type constant current peristaltic pump and the 100KD of the Millipore company hollow-fibre membrane post in Shanghai to carry out ultrafiltration, circulate 5 times, circular buffering liquid uses 20mM, the phosphate buffered saline buffer of pH7.0, the final collection appeared liquid 80mL, supernatant liquor total protein concentration 3.8g/L, people source a-whey albumin concentration is 1.2g/L, and ox source a-whey albumin concentration is 0.8g/L.
Use 20mM, the pH6.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 1.2M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 6.0, sample introduction is to using 20mM, pH6.0 contains in the good Phenyl Sepharose 6FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 1.2M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GEAKTAExplorer100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH6.0 to contain 1.2M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 155mL in the collection, total protein concentration is 1.0g/L, and people source a-whey albumin concentration is 0.3g/L, and ox source a-whey albumin concentration is 0.3g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH6.0 is also collected elution peak 105mL, and total protein concentration is 1.0g/L, and people source a-whey albumin concentration is 0.5g/L, purity 50%, and the technology rate of recovery 37.5%, ox source a-whey albumin concentration is 0.2g/L.
Get 100mL transgenic human source a-whey albumin cow's milk, the cow's milk total protein concentration is 33g/L, people source a-whey albumin concentration is 1.4g/L, ox source a-whey albumin concentration is 1.0g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.5g/L, people source a-whey albumin concentration is 1.7g/L, and ox source a-whey albumin concentration is 1.2g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 9.0, use the DDB-300 of the letter Instr Ltd. type constant current peristaltic pump and the 150KD of the Millipore company hollow-fibre membrane post in Shanghai to carry out ultrafiltration, circulate 5 times, circular buffering liquid uses 20mM, the phosphate buffered saline buffer of pH9.0, the final collection appeared liquid 85mL, supernatant liquor total protein concentration 4.6g/L, people source a-whey albumin concentration is 1.3g/L, and ox source a-whey albumin concentration is 0.9g/L.
Use 20mM, the pH7.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 1.0M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 7.0, sample introduction is to using 20mM, pH7.0 contains in the good Octyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 1.0M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GEAKTAExplorer 100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH7.0 to contain 1.0M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 165mL in the collection, total protein concentration is 1.4g/L, and people source a-whey albumin concentration is 0.01g/L, and ox source a-whey albumin concentration is 0.3g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH7.0 is also collected elution peak 120mL, and total protein concentration is 1.2g/L, and people source a-whey albumin concentration is 0.9g/L, purity 75%, and the technology rate of recovery 77%, ox source a-whey albumin concentration is 0.2g/L.
Embodiment 5
Get 100mL transgenic human source a-whey albumin cow's milk, the cow's milk total protein concentration is 33g/L, people source a-whey albumin concentration is 1.4g/L, ox source a-whey albumin concentration is 1.0g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.5g/L, people source a-whey albumin concentration is 1.7g/L, and ox source a-whey albumin concentration is 1.2g/L.
Use the ammonium sulfate powder to regulate the whey sample ammonium sulfate concentrations and reach 0.8M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 8.0, sample introduction is to using 20mM, pH8.0 contains in the good Octyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 0.8M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GE AKTA Explorer 100, ultraviolet 280nm wavelength absorption detects, flow velocity 10ml/min, after finishing, last sample uses 20mM, the phosphate buffered saline buffer that pH8.0 contains 0.8M ammonium sulfate continues to wash to 280nm uv-absorbing signal gets back to baseline, in the collection sample penetrate peak 150mL, total protein concentration is 3.8g/L, people source a-whey albumin concentration is 0.2g/L, and ox source a-whey albumin concentration is 0.5g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH8.0 is also collected elution peak 112mL, and total protein concentration is 1.5g/L, and people source a-whey albumin concentration is 0.9g/L, and ox source a-whey albumin concentration is 0.2g/L.
Get elution peak 30mL sample introduction to using 20mM, pH7.0 contains in the good Superdex 75 gel permeation chromatography posts of the phosphate buffered saline buffer pre-equilibration of 0.1M sodium sulfate, 3.5 centimetres of column internal diameters, 60 centimetres of post bed heights, tomography devices GE AKTA Explorer 100, ultraviolet 280nm wavelength absorption detects, flow velocity 2mL/min, the phosphate buffered saline buffer that continues after last sample finishes to use 20mM, pH7.0 to contain 0.1M sodium sulfate washes, and the protein peak of collecting the 330mL place is total to 80mL, total protein concentration is 0.35g/L, people source a-whey albumin concentration is 0.3g/L, purity 86%, the technology rate of recovery 17%.
Embodiment 6
Get 100mL transgenic human source Lactotransferrin cow's milk, the cow's milk total protein concentration is 34g/L, people source Lactotransferrin concentration is 1.7g/L, ox source Lactotransferrin concentration is 0.1g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 98g/L, people source Lactotransferrin concentration is 2.0g/L, and ox source Lactotransferrin concentration is 0.1g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 9.0, slowly adding the ammonium sulfate powder is 50% to whey ammonium sulfate saturation ratio, with the mole numeration is 2.37M, after at room temperature vibrating 2 hours, deposit sample is used under 4 degrees centigrade in the Sigma 3k30 whizzer, and to leave mental and physical efforts under order be centrifugal 30 minutes of 10000g and collect supernatant liquor 75mL, supernatant liquor total protein concentration 3.6g/L, people source Lactotransferrin concentration is 1.5g/L, and ox source Lactotransferrin concentration is 0.1g/L.
Use 20mM, the pH6.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 1.0M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 6.0, sample introduction is to using 20mM, pH6.0 contains in the good Butyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 1.0M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GE AKTA Explorer 100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH6.0 to contain 1.0M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 148mL in the collection, total protein concentration is 1.6g/L, and people source Lactotransferrin concentration is 0.2g/L, and ox source Lactotransferrin concentration is 0.05g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH6.0 is also collected elution peak 103mL, and total protein concentration is 0.9g/L, and people source Lactotransferrin concentration is 0.8g/L, purity 89%, and the technology rate of recovery 48%, ox source Lactotransferrin concentration is 0.001g/L.
Embodiment 7
Get 100mL transgenic human source Lactotransferrin cow's milk, the cow's milk total protein concentration is 34g/L, people source Lactotransferrin concentration is 1.7g/L, ox source Lactotransferrin concentration is 0.1g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.8g/L, people source Lactotransferrin concentration is 2.0g/L, and ox source Lactotransferrin concentration is 0.1g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 8.0, use the DDB-300 of the letter Instr Ltd. type constant current peristaltic pump and the 10KD of the Millipore company hollow-fibre membrane post in Shanghai to carry out ultrafiltration, circulate 5 times, circular buffering liquid uses 20mM, the phosphate buffered saline buffer of pH8.0, the final collection appeared liquid 77mL, supernatant liquor total protein concentration 4.2g/L, people source Lactotransferrin concentration is 1.4g/L, and ox source Lactotransferrin concentration is 0.1g/L.
Use 20mM, the pH7.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 0.6M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 6.0, sample introduction is to using 20mM, pH7.0 contains in the good Octyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 0.6M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GE AKTA Explorer100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH6.0 to contain 0.6M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 152mL in the collection, total protein concentration is 1.4g/L, and people source Lactotransferrin concentration is 0.3g/L, and ox source Lactotransferrin concentration is 0.04g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH7.0 is also collected elution peak 106mL, and total protein concentration is 1.0g/L, and people source Lactotransferrin concentration is 0.5g/L, purity 50%, and the technology rate of recovery 31%, ox source Lactotransferrin concentration is 0.02g/L.
Embodiment 8
Get 100mL transgenic human source N,O-Diacetylmuramidase cow's milk, the cow's milk total protein concentration is 32g/L, people source lysozyme concentration is 1.5g/L, ox source lysozyme concentration is 0.01g/L, under 4 degrees centigrade in the use Sigma 3k30 whizzer, leaving mental and physical efforts under order is to collect supernatant liquor acquisition whey 80mL after 10000g removed lipid and casein in centrifugal 30 minutes, and total protein concentration is 9.3g/L, people source lysozyme concentration is 1.7g/L, and ox source lysozyme concentration is 0.01g/L.Use hydrochloric acid or the sodium hydroxide solution of 1M to regulate whey pH to 8.0, use the DDB-300 of the letter Instr Ltd. type constant current peristaltic pump and the 150KD of the Millipore company hollow-fibre membrane post in Shanghai to carry out ultrafiltration, circulate 5 times, circular buffering liquid uses 20mM, the phosphate buffered saline buffer of pH8.0, the final collection appeared liquid 85mL, supernatant liquor total protein concentration 3.4g/L, people source a-whey albumin concentration is 1.4g/L, and ox source a-whey albumin concentration is 0.008g/L.
Use 20mM, the pH8.0 phosphate buffered saline buffer is regulated whey pretreatment sample ammonium sulfate concentrations and is reached 0.9M, use 1M hydrochloric acid or sodium hydroxide solution to regulate whey pretreatment sample pH to 8.0, sample introduction is to using 20mM, pH8.0 contains in the good Octyl Sepharose 4FF hydrophobic chromatography post of the phosphate buffered saline buffer pre-equilibration of 0.9M ammonium sulfate, 3.5 centimetres of column internal diameters, 10 centimetres of post bed heights, tomography devices GE AKTA Explorer100, ultraviolet 280nm wavelength absorption detects, finish phosphate buffered saline buffer that the back uses 20mM, pH8.0 to contain 0.9M ammonium sulfate of flow velocity 10ml/min, last sample continues to wash to 280nm uv-absorbing signal and gets back to baseline, sample penetrates peak 150mL in the collection, total protein concentration is 1.1g/L, and people source lysozyme concentration is 0.35g/L, and ox source lysozyme concentration is 0.004g/L; Use 20mM again, the phosphate buffered saline buffer one-step elution of pH8.0 is also collected elution peak 112mL, and total protein concentration is 0.8g/L, and people source lysozyme concentration is 0.5g/L, purity 62.5%, and the technology rate of recovery 37%, ox source lysozyme concentration is 0.0001g/L.
Claims (7)
1. the separation purification method of heterogenous homogeneous serum albumin, its step comprises:
1) choosing the transgene cow milk that contains at least a people source whey-protein is raw material, collects supernatant liquor behind centrifugal removal lipid and the casein and obtains whey;
2) the gained whey is removed macromole impurity such as immunoglobulin (Ig), remaining casein through pre-treatment;
3), separate obtaining heterogenous homogeneous serum albumin with the combination chromatography or the single step hydrophobic interaction chromatography of pretreated whey through hydrophobic interaction chromatography and gel permeation chromatography coupling;
Described allos behaviour source and Niu Yuan, described whey-protein are alpha-whey albumin, Lactotransferrin and N,O-Diacetylmuramidase.
2. the method for claim 1, pre-treatment step is ammonium sulfate precipitation method or uf processing method.
3. the method for claim 1, hydrophobic chromatoghaphy medium is an agarose matrix, its aglucon is a butyl.
4. the method for claim 1, the used salt of hydrophobic chromatography is ammonium sulfate, its concentration is 0.5-2M ammonium sulfate.
5. the method for claim 1, the used buffer system of hydrophobic chromatography is a phosphate buffered saline buffer.
6. the method for claim 1, hydrophobic chromatography single treatment amount is the cow's milk raw material of 2 times of column volumes.
7. the method for claim 1, the gel permeation chromatography medium is Superdex 75.
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| CN101948529B (en) * | 2010-08-13 | 2013-05-01 | 中国科学院过程工程研究所 | Hydrophobic chromatography separating and purifying method for efficiently purifying metal ion binding proteins |
| CN102286096A (en) * | 2011-07-08 | 2011-12-21 | 上海交通大学 | Scale purification method for lactalbumin |
| EP3179862A1 (en) * | 2014-08-15 | 2017-06-21 | Upfront Chromatography A/S | Method for separating alpha-lactalbumin and beta-lactoglobulin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996002573A1 (en) * | 1994-07-20 | 1996-02-01 | Pharming Bv | Separation of human serum albumin |
| CN1661018A (en) * | 2004-10-11 | 2005-08-31 | 中国人民解放军第四军医大学 | Carrier for expressing promotor of casein gene of milch goats grew up in the entral shanxi plain |
| CN1850974A (en) * | 2005-04-21 | 2006-10-25 | 李宁 | Human alpha-lacto albumin gene transgenic cloned macro domectic animal production method |
| CN101104635A (en) * | 2007-04-30 | 2008-01-16 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996002573A1 (en) * | 1994-07-20 | 1996-02-01 | Pharming Bv | Separation of human serum albumin |
| CN1661018A (en) * | 2004-10-11 | 2005-08-31 | 中国人民解放军第四军医大学 | Carrier for expressing promotor of casein gene of milch goats grew up in the entral shanxi plain |
| CN1850974A (en) * | 2005-04-21 | 2006-10-25 | 李宁 | Human alpha-lacto albumin gene transgenic cloned macro domectic animal production method |
| CN101104635A (en) * | 2007-04-30 | 2008-01-16 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
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