CN107903301A - A kind of separation method for glycosylating albumen - Google Patents
A kind of separation method for glycosylating albumen Download PDFInfo
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- CN107903301A CN107903301A CN201711415528.5A CN201711415528A CN107903301A CN 107903301 A CN107903301 A CN 107903301A CN 201711415528 A CN201711415528 A CN 201711415528A CN 107903301 A CN107903301 A CN 107903301A
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- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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Abstract
The invention discloses a kind of separation method for glycosylating albumen, belong to protein separation technology field；The separation method of albumen is glycosylated, is comprised the following steps：Sample separation, isolated glycosylation albumen crude product are carried out using reversed-phase high performance liquid chromatography；Wherein, the chromatographic buffers of sample dissolving contain Ca2+、NH4 +, Ca in chromatographic buffers2+Concentration be 3~4mM, NH in the chromatographic buffers4 +Concentration be 12~20mM；Beads enrichment：Glycosylation albumen crude product is further purified, obtains glycosylation albumen sterling.The separation method of the present invention can improve the separation purity of glycosylation albumen.
Description
Technical field
The invention belongs to protein separation technology field, more particularly to a kind of separation method for glycosylating albumen.
Background technology
In protein production process, the separation for glycosylating albumen is a unique research field.Glycosylate in albumen
Contain impurity or non-glycosylated protein, it is necessary to which impurity and non-glycosylated protein are removed.Existing separation method is more
Single, accurate not high enough, such as reversed-phased high performace liquid chromatographic, electrophoresis and ion exchange chromatography.
Therefore, for glycosylation albumen accuracy it is low the problem of, it is necessary to develop a kind of new glycosylation albumen point
From method, to improve the separation purity of glycosylation albumen.
The content of the invention
Present invention aims to overcome that the shortcomings of the prior art, and a kind of separation method for glycosylating albumen is provided,
With the separation purity of the glycosylation albumen of raising.
To achieve the above object, the technical solution taken of the present invention is：A kind of separation method for glycosylating albumen, it includes
Following steps：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography；Wherein, sample
The chromatographic buffers of dissolving contain Ca2+、NH4 +, Ca in the chromatographic buffers2+Concentration be 3~4mM, the chromatographic buffers
Middle NH4 +Concentration be 12~20mM.
S2) Beads enrichment：The glycosylation albumen crude product is further purified, obtains glycosylation albumen sterling.
The present invention first passes through reversed-phase high performance liquid chromatography separation glycosylation albumen, obtains glycosylation albumen crude product；Then again
Beads enrichment is carried out to glycosylation albumen crude product, further isolated glycosylation albumen sterling；Reversed-phase high performance liquid chromatography point
Complement each other from Beads enrichment, collaboration improves the separation purity of glycosylation albumen.In reversed-phase high performance liquid chromatography, chromatography is delayed
Contain 3~4mM Ca in fliud flushing2+With 12~20mM NH4 +, can will glycosylation albumen and non-glycosylated protein separate, with up to
To more preferable separating effect.
As the improvement of above-mentioned technical proposal, in step S1) in, the pH value of the chromatographic buffers is 7.5~8, chromatography
Separated chromatography temperature is 26~30 DEG C.
As the improvement of above-mentioned technical proposal, the Ca2+Come from calcium chloride or calcium nitrate, the NH4 +Come from chlorination
Ammonium or ammonium nitrate.
As the improvement of above-mentioned technical proposal, the Beads enrichment comprises the following steps：
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing
Magnetic Isolation, agglutinin solution is added after abandoning supernatant, 35~37 DEG C of 1~2h of reaction, supernatant discarding into container again to obtain the final product
To epoxy ethyl derivatization magnetic bead-agglutinin compound；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and is discarded
Clear liquid；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, shakes at room temperature
Shake 4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
During Beads enrichment purifying glycosylation protein, the present invention first builds epoxy ethyl derivatization magnetic bead-agglutinin
Compound, builds epoxy ethyl derivatization magnetic bead-agglutinin-glycosylation albumen composition with glycosylation albumen again afterwards, then uses
Albumen is glycosylated under elution；The cleaning buffer solution and eluent used during this fully cooperates with, it is ensured that isolated
Glycosylation albumen purity it is high.
Further as above-mentioned technical proposal is improved, and the coupling buffer is to contain 0.2~0.3M saleratus and 1
~2M sodium chloride.
Further as above-mentioned technical proposal is improved, and the confining liquid is the acetate buffer containing bovine serum albumin
Liquid.
Further as above-mentioned technical proposal is improved, and the combination buffer is the sodium phosphate buffer containing polysorbas20
Liquid, and the mass concentration of sodium phosphate is 1%~2% in the sodium phosphate buffer.
Further as above-mentioned technical proposal is improved, the cleaning buffer solution I be containing 0.5~0.9mM calcium chloride,
The Tris-HCl buffer solutions of 1.1~1.5mM manganese chlorides.
Further as above-mentioned technical proposal is improved, and the cleaning buffer solution II is to contain 1.2~2mM potassium chloride, 0.6
The Tris-HCl buffer solutions of~1.3mM sodium chloride.
Further as above-mentioned technical proposal is improved, the eluent be containing 1.2~2mM potassium chloride, 0.6~
The Tris-HCl buffer solutions of 1.3mM sodium chloride and 40~60mM Alpha-Methyl mannoses.
The beneficial effects of the present invention are：The present invention provides a kind of separation method for glycosylating albumen, and the present invention first passes through
Reversed-phase high performance liquid chromatography separation glycosylation albumen, obtains glycosylation albumen crude product；Then glycosylation albumen crude product is carried out again
Beads enrichment, it is further isolated to glycosylate albumen sterling；Reversed-phase high performance liquid chromatography separates and Beads enrichment complements each other,
Collaboration improves the separation purity of glycosylation albumen；In reversed-phase high performance liquid chromatography, 3~4mM Ca are contained in chromatographic buffers2+
With 12~20mM NH4 +, glycosylation albumen and non-glycosylated protein can be separated, to reach more preferable separating effect；Secondly
The cleaning buffer solution and eluent used during Beads enrichment fully cooperates with, it is ensured that the purity of isolated glycosylation albumen
It is high.
Embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
The present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose), successively including following step
Suddenly：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography
Reversed-phase high performance liquid chromatography condition：The filler of chromatographic column is C18Reverse phase silica gel particle, chromatographic column size are 10 μ ms
250mm, Detection wavelength 280nm, the temperature of chromatographic isolation chromatography is 26 DEG C, and mobile phase is to contain 40mM Tris and 100mM
75% ethanol water of KCl；Sample is dissolved in Tris-HCl buffer solutions, the pH value of Tris-HCl buffer solutions is 7.5, and
Tris-HCl buffer solutions contain 3mM Ca2+With 12mM NH4 +；Wherein, Ca2+Come from calcium chloride, the NH4 +Come from nitric acid
Ammonium；
S2 Beads enrichment) is carried out to glycosylation albumen crude product
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing
Magnetic Isolation, agglutinin solution is added after abandoning supernatant, 35~37 DEG C of 1~2h of reaction, supernatant discarding into container again to obtain the final product
To epoxy ethyl derivatization magnetic bead-agglutinin compound；Wherein, the coupling buffer is to contain 0.2M saleratus and 1M chlorine
Change sodium；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；Wherein, clean
Buffer solution I is the Tris-HCl buffer solutions containing 0.5mM calcium chloride, 1.1mM manganese chlorides；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and is discarded
Clear liquid；Wherein, confining liquid is the sodium acetate buffer containing bovine serum albumin；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, shakes at room temperature
Shake 4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；Wherein, combination buffer is the phosphoric acid containing polysorbas20
Sodium buffer solution, and the mass concentration of sodium phosphate is 1% in the sodium phosphate buffer；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；Wherein, cleaning buffer solution II is the Tris-HCl bufferings containing 1.2mM potassium chloride, 0.6mM sodium chloride
Liquid；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；Wherein, eluent be containing 1.2mM potassium chloride, 0.6mM sodium chloride and
The Tris-HCl buffer solutions of 40mM Alpha-Methyl mannoses；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
Embodiment 2
The present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose), successively including following step
Suddenly：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography
Reversed-phase high performance liquid chromatography condition：The filler of chromatographic column is C18Reverse phase silica gel particle, chromatographic column size are 10 μ ms
250mm, Detection wavelength 280nm, the temperature of chromatographic isolation chromatography is 28 DEG C, and mobile phase is to contain 40mM Tris and 100mM
75% ethanol water of KCl；Sample is dissolved in Tris-HCl buffer solutions, the pH value of Tris-HCl buffer solutions is 7.7, and
Tris-HCl buffer solutions contain 3.2mM Ca2+With 13.6mM NH4 +；Wherein, Ca2+Come from calcium chloride, the NH4 +Come from chlorine
Change ammonium；
S2 Beads enrichment) is carried out to glycosylation albumen crude product
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing
Magnetic Isolation, agglutinin solution is added after abandoning supernatant, 35~37 DEG C of 1~2h of reaction, go supernatant to obtain into container again
Epoxy ethyl derivatization magnetic bead-agglutinin compound；Wherein, the coupling buffer is to contain 0.22M saleratus and 1.4M
Sodium chloride；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；Wherein, clean
Buffer solution I is the Tris-HCl buffer solutions containing 0.6mM calcium chloride, 1.2mM manganese chlorides；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and is discarded
Clear liquid；Wherein, confining liquid is the sodium acetate buffer containing bovine serum albumin；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, shakes at room temperature
Shake 4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；Wherein, combination buffer is the phosphoric acid containing polysorbas20
Sodium buffer solution, and the mass concentration of sodium phosphate is 1.4% in the sodium phosphate buffer；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；Wherein, cleaning buffer solution II is the Tris-HCl bufferings containing 1.4mM potassium chloride, 0.9mM sodium chloride
Liquid；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；Wherein, eluent be containing 1.5mM potassium chloride, 0.7mM sodium chloride and
The Tris-HCl buffer solutions of 48mM Alpha-Methyl mannoses；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
Embodiment 3
The present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose), successively including following step
Suddenly：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography
Reversed-phase high performance liquid chromatography condition：The filler of chromatographic column is C18Reverse phase silica gel particle, chromatographic column size are 10 μ ms
250mm, Detection wavelength 280nm, the temperature of chromatographic isolation chromatography is 29 DEG C, and mobile phase is to contain 40mM Tris and 100mM
75% ethanol water of KCl；Sample is dissolved in Tris-HCl buffer solutions, the pH value of Tris-HCl buffer solutions is 7.9, and
Tris-HCl buffer solutions contain 3.7mM Ca2+With 16mM NH4 +；Wherein, Ca2+Come from calcium nitrate, the NH4 +Come from nitric acid
Ammonium；
S2 Beads enrichment) is carried out to glycosylation albumen crude product
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing
Magnetic Isolation, agglutinin solution is added after abandoning supernatant, 35~37 DEG C of 1~2h of reaction, supernatant discarding into container again to obtain the final product
To epoxy ethyl derivatization magnetic bead-agglutinin compound；Wherein, the coupling buffer be containing 0.28M saleratus and
1.65M sodium chloride；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；Wherein, clean
Buffer solution I is the Tris-HCl buffer solutions containing 0.8mM calcium chloride, 1.4mM manganese chlorides；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and is discarded
Clear liquid；Wherein, confining liquid is the sodium acetate buffer containing bovine serum albumin；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, shakes at room temperature
Shake 4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；Wherein, combination buffer is the phosphoric acid containing polysorbas20
Sodium buffer solution, and the mass concentration of sodium phosphate is 1.7% in the sodium phosphate buffer；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；Wherein, cleaning buffer solution II is the Tris-HCl bufferings containing 1.7mM potassium chloride, 1.2mM sodium chloride
Liquid；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；Wherein, eluent is to contain 1.8mM potassium chloride, 1mM sodium chloride and 52mM
The Tris-HCl buffer solutions of Alpha-Methyl mannose；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
Embodiment 4
The present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose), successively including following step
Suddenly：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography
Reversed-phase high performance liquid chromatography condition：The filler of chromatographic column is C18Reverse phase silica gel particle, chromatographic column size are 10 μ ms
250mm, Detection wavelength 280nm, the temperature of chromatographic isolation chromatography is 30 DEG C, and mobile phase is to contain 40mM Tris and 100mM
75% ethanol water of KCl；Sample is dissolved in Tris-HCl buffer solutions, the pH value of Tris-HCl buffer solutions is 8, and
Tris-HCl buffer solutions contain 4mM Ca2+With 20mM NH4 +；Wherein, Ca2+Come from calcium nitrate, the NH4 +Come from nitric acid
Ammonium；
S2 Beads enrichment) is carried out to glycosylation albumen crude product
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing
Magnetic Isolation, agglutinin solution is added after abandoning supernatant, 35~37 DEG C of 1~2h of reaction, supernatant discarding into container again to obtain the final product
To epoxy ethyl derivatization magnetic bead-agglutinin compound；Wherein, the coupling buffer is to contain 0.3M saleratus and 2M chlorine
Change sodium；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；Wherein, clean
Buffer solution I is the Tris-HCl buffer solutions containing 0.9mM calcium chloride, 1.5mM manganese chlorides；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and is discarded
Clear liquid；Wherein, confining liquid is the sodium acetate buffer containing bovine serum albumin；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, shakes at room temperature
Shake 4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；Wherein, combination buffer is the phosphoric acid containing polysorbas20
Sodium buffer solution, and the mass concentration of sodium phosphate is 2% in the sodium phosphate buffer；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；Wherein, cleaning buffer solution II is the Tris-HCl buffer solutions containing 2mM potassium chloride, 1.3mM sodium chloride；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；Wherein, eluent is to contain 2mM potassium chloride, 1.3mM sodium chloride and 60mM
The Tris-HCl buffer solutions of Alpha-Methyl mannose；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
Comparative example 1
The present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose), using reversed phase high performance liquid
Phase chromatography carries out sample and isolates and purifies：
Reversed-phase high performance liquid chromatography condition：The filler of chromatographic column is C18Reverse phase silica gel particle, chromatographic column size are 10 μ ms
250mm, Detection wavelength 280nm, the temperature of chromatographic isolation chromatography is 30 DEG C, and mobile phase is to contain 40mM Tris and 100mM
75% ethanol water of KCl；Sample is dissolved in Tris-HCl buffer solutions, the pH value of Tris-HCl buffer solutions is 8, and
Tris-HCl buffer solutions contain 4mM Ca2+With 20mM NH4 +；Wherein, Ca2+Come from calcium nitrate, the NH4 +Come from nitric acid
Ammonium.
Comparative example 2
It is thick to glycosylation albumen the present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose)
Product carry out Beads enrichment：
S1) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, magnetic is carried out after mixing
Property separation, add agglutinin solution after abandoning supernatant into container again, 35~37 DEG C of 1~2h of reaction go supernatant to obtain ring
Oxygen derivatized magnetic bead-agglutinin compound；Wherein, the coupling buffer is to contain 0.3M saleratus and 2M chlorinations
Sodium；
S2 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；Wherein, clean
Buffer solution I is the Tris-HCl buffer solutions containing 0.9mM calcium chloride, 1.5mM manganese chlorides；
S3 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and removes supernatant
Liquid；Wherein, confining liquid is the sodium acetate buffer containing bovine serum albumin；
S4 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S5 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, rocks at room temperature
4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；Wherein, combination buffer is the sodium phosphate containing polysorbas20
Buffer solution, and the mass concentration of sodium phosphate is 2% in the sodium phosphate buffer；
S6 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；Wherein, cleaning buffer solution II is the Tris-HCl buffer solutions containing 2mM potassium chloride, 1.3mM sodium chloride；
S7 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S8 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；Wherein, eluent is to contain 2mM potassium chloride, 1.3mM sodium chloride and 60mM
The Tris-HCl buffer solutions of Alpha-Methyl mannose；
S9) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
Comparative example 3
The present embodiment provides a kind of separation method of glycosylated insulin (N-terminal connection mannose), successively including following step
Suddenly：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography
Reversed-phase high performance liquid chromatography condition：The filler of chromatographic column is C18Reverse phase silica gel particle, chromatographic column size are 10 μ ms
250mm, Detection wavelength 280nm, the temperature of chromatographic isolation chromatography is 30 DEG C, and mobile phase is to contain 40mM Tris and 100mM
75% ethanol water of KCl；Sample is dissolved in Tris-HCl buffer solutions, the pH value of Tris-HCl buffer solutions is 8, and
Tris-HCl buffer solutions contain 1mM Ca2+With 10mM NH4 +；Wherein, Ca2+Come from calcium nitrate, the NH4 +Come from nitric acid
Ammonium；
S2 Beads enrichment) is carried out to glycosylation albumen crude product
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing
Magnetic Isolation, agglutinin solution is added after abandoning supernatant, 35~37 DEG C of 1~2h of reaction, go supernatant to obtain into container again
Epoxy ethyl derivatization magnetic bead-agglutinin compound；Wherein, the coupling buffer is to contain 0.3M saleratus and 2M chlorinations
Sodium；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；Wherein, clean
Buffer solution I is the Tris-HCl buffer solutions containing 8mM calcium chloride, 6mM manganese chlorides；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and removes supernatant
Liquid；Wherein, confining liquid is the sodium acetate buffer containing bovine serum albumin；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, the glycosylation albumen crude product and ultra-pure water is added, shakes at room temperature
Shake 4~6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；Wherein, combination buffer is the phosphoric acid containing polysorbas20
Sodium buffer solution, and the mass concentration of sodium phosphate is 2% in the sodium phosphate buffer；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；Wherein, cleaning buffer solution II is the Tris-HCl buffer solutions containing 2mM potassium chloride；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, magnetic point is carried out
From, and abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out,
Supernatant is taken, up to albumen sterling solution is glycosylated；Wherein, eluent is to contain 2mM sodium chloride and 60mM Alpha-Methyl mannoses
Tris-HCl buffer solutions；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, albumen is pure up to glycosylating
Product.
Effect example
The separation purity of 1~3 glycosylated insulin of embodiment 1~4 and comparative example is measured, assay method uses reversed phase high efficiency
Liquid chromatogram, refers to the reversed-phase high performance liquid chromatography in embodiment 3, the purity testing result such as institute of table 1 of glycosylated insulin
Show.
Table 1
| Group | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Purity of protein % | 94.1 | 97.3 | 97.6 | 95 | 78.2 | 83.3 | 86.9 |
As shown in table 1, separation method using the present invention, isolating and purifying for glycosylated insulin are better；Thus,
It can be seen that reversed-phase high performance liquid chromatography separation and Beads enrichment complement each other, collaboration improves the purity of separated glycosylation albumen；
Added in reversed-phase high performance liquid chromatography, in chromatographic buffers and contain 3~4mM Ca2+With 12~20mM NH4 +, will can glycosylate
Albumen and non-glycosylated protein separate, to reach more preferable separating effect；Secondly the cleaning used during Beads enrichment is delayed
Fliud flushing and eluent fully cooperate with, it is ensured that the purity of isolated glycosylation albumen is high
Finally, it should be noted that above example to illustrate technical scheme rather than to the present invention protect
The limitation of scope, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should manage
Solution, can modify to technical scheme or replace on an equal basis, without departing from technical solution of the present invention essence and
Scope.
Claims (10)
1. a kind of separation method for glycosylating albumen, it is characterised in that comprise the following steps：
S1 sample separation, isolated glycosylation albumen crude product) are carried out using reversed-phase high performance liquid chromatography；Wherein, sample dissolves
Chromatographic buffers contain Ca2+、NH4 +, Ca in the chromatographic buffers2+Concentration be 3~4mM, in the chromatographic buffers
NH4 +Concentration be 12~20mM.
S2) Beads enrichment：The glycosylation albumen crude product is further purified, obtains glycosylation albumen sterling.
2. separation method as claimed in claim 1, it is characterised in that in step S1) in, the pH value of the chromatographic buffers is
7.5~8, the chromatography temperature of chromatographic isolation is 26~30 DEG C.
3. separation method as claimed in claim 1, it is characterised in that the Ca2+Come from calcium chloride or calcium nitrate, it is described
NH4 +Come from ammonium chloride or ammonium nitrate.
4. separation method as claimed in claim 1, it is characterised in that the Beads enrichment comprises the following steps：
S21) epoxy ethyl derivatization magnetic bead is added in container, coupling buffer is added into container, is carried out after mixing magnetic
Separation, supernatant add agglutinin solution, 35~37 DEG C of 1~2h of reaction, abandoning supernatant into container again after discarding；
S22 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S23 confining liquid, 35~37 DEG C of 1~2h of reaction) are added into container；Magnetic Isolation is carried out after reaction, and is discarded
Clear liquid；
S24 cleaning buffer solution I) is added into container, Magnetic Isolation, and abandoning supernatant are carried out after mixing；
S25 combination buffer) is added into container, adds the glycosylation albumen crude product and ultra-pure water, rock 4 at room temperature~
6h；After rocking, Magnetic Isolation, abandoning supernatant are carried out；
S26 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, Magnetic Isolation is carried out, and
Abandoning supernatant；
S27 cleaning buffer solution II) is added into container, rocks 1~3min at room temperature；After rocking, Magnetic Isolation is carried out, and
Abandoning supernatant；
S28 eluent, 25~29 DEG C of 25~30min of elution) are added into container；After elution, Magnetic Isolation is carried out, is taken
Clear liquid, up to albumen sterling solution is glycosylated；
S29) the glycosylation albumen sterling solution is dialysed, finally sloughs eluent, up to albumen sterling is glycosylated.
5. separation method as claimed in claim 4, it is characterised in that the coupling buffer contains 0.2~0.3M bicarbonates
Potassium and 1~2M sodium chloride.
6. separation method as claimed in claim 4, it is characterised in that the confining liquid is the sodium acetate containing bovine serum albumin
Buffer solution.
7. separation method as claimed in claim 4, it is characterised in that the combination buffer is the sodium phosphate containing polysorbas20
Buffer solution, and the mass concentration of sodium phosphate is 1%~2% in the sodium phosphate buffer.
8. separation method as claimed in claim 4, it is characterised in that the cleaning buffer solution I is to contain 0.5~0.9mM chlorine
Change calcium, the Tris-HCl buffer solutions of 1.1~1.5mM manganese chlorides.
9. separation method as claimed in claim 4, it is characterised in that the cleaning buffer solution II is to contain 1.2~2mM chlorinations
The Tris-HCl buffer solutions of potassium, 0.6~1.3mM sodium chloride.
10. separation method as claimed in claim 4, it is characterised in that the eluent be containing 1.2~2mM potassium chloride,
The Tris-HCl buffer solutions of 0.6~1.3mM sodium chloride and 40~60m M Alpha-Methyl mannoses.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110846295A (en) * | 2019-11-13 | 2020-02-28 | 上海交通大学 | Method for enriching potato acyl hydrolase based on functional nano magnetic beads |
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| US6180757B1 (en) * | 1998-04-15 | 2001-01-30 | Novo Nordisk A/S | Process for the separation of proteins using a CA++ containing eluant |
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| KRISHNAMURTHY VENKATESAN ET AL: "A STUDY ON INTERACTION OF ION-PAIRING AGENT IN SEPARATION OF HUMAN INSULIN AND GLYCOSYLATED HUMAN INSULIN USING REVERSE PHASE LIQUID CHROMATOGRAPHY", 《JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES》 * |
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| CN110846295A (en) * | 2019-11-13 | 2020-02-28 | 上海交通大学 | Method for enriching potato acyl hydrolase based on functional nano magnetic beads |
| CN110846295B (en) * | 2019-11-13 | 2022-12-23 | 上海交通大学 | Method for enriching potato acyl hydrolase based on functional nano magnetic beads |
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