CN114939400A - Affinity chromatography filler, preparation method thereof and affinity purification process - Google Patents

Affinity chromatography filler, preparation method thereof and affinity purification process Download PDF

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CN114939400A
CN114939400A CN202210749834.7A CN202210749834A CN114939400A CN 114939400 A CN114939400 A CN 114939400A CN 202210749834 A CN202210749834 A CN 202210749834A CN 114939400 A CN114939400 A CN 114939400A
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钱剩金
陈丽
杨侠斐
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Shanghai Zhaowei Bioengineering Co ltd
Shanghai Hongene Biotech Corp
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Abstract

The application relates to the technical field of biological macromolecule separation and purification, in particular to an affinity chromatography filler, a preparation method thereof and an affinity purification process. The affinity chromatography packing comprises an activated support and a ligand attached to the activated support. The affinity chromatography packing provided by the application can effectively separate and purify the mutant T7RNA polymerase shown by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 in the amino acid sequence of the target protein by connecting the ligand containing a locked nucleic acid structure and an amino group to the activated carrier, so that the yield and the purity of the purified target protein are effectively improved.

Description

Affinity chromatography filler, preparation method thereof and affinity purification process
Technical Field
The application relates to the technical field of biological macromolecule separation and purification, in particular to an affinity chromatography filler, a preparation method thereof and an affinity purification process.
Background
The mutant T7RNA polymerase with the amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 can be used for transcription synthesis of RNA, and the preparation of the mutant T7RNA polymerase with the amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 generally comprises the steps of designing a codon primer containing a corresponding amino acid mutation site, amplifying a gene sequence of the T7RNA polymerase containing a correct mutation sequence through polymerase chain reaction in a PCR reaction system, and expressing the mutant T7RNA polymerase required for thallus expression.
However, the expression system for expressing the bacteria is complex, and the expressed polymerase not only comprises the required mutated T7RNA polymerase with the amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7, but also comprises other non-target proteins which are not well expressed. At present, the purification method for purifying the system to obtain the mutant T7RNA polymerase shown by the amino acid sequence of the target protein such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 is complex, and the yield and the purity of the target protein are not high, so that the industrial amplification production and the further application of the target protein are not facilitated.
Disclosure of Invention
The application aims to provide an affinity chromatography filler, a preparation method thereof and an affinity purification process, and aims to solve the technical problems that the yield and the purity of the mutant T7RNA polymerase shown by an amino acid sequence of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7 are not high in the prior art.
The first aspect of the application provides an affinity chromatography filler, which comprises an activated carrier and a ligand connected to the activated carrier; the structural formula of the ligand is as follows:
Figure BDA0003717917650000021
wherein B is a natural or modified amino-containing nucleobase.
The affinity chromatography packing provided by the application can effectively separate and purify T7RNA polymerase of which the amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7, and effectively improves the yield and purity of the purified target protein by connecting the ligand containing a locked nucleic acid structure and an amino group to the activated carrier.
In a second aspect, the present application provides a method for preparing an affinity chromatography packing, comprising a coupling reaction of the ligand mentioned in the first aspect with an activated carrier; the preparation method of the activated carrier comprises the step of activating the carrier by using an activating agent.
The affinity chromatography filler prepared by the method can be used for purifying and separating mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 of the amino acid sequence of the target protein, and can effectively improve the yield and purity of the purified target protein.
In a third aspect, the present application provides an affinity purification process, comprising performing purification treatment on a system containing a target protein by using an affinity chromatography column on which the affinity chromatography packing provided in the first aspect is placed; the target protein comprises mutant T7RNA polymerase, and the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7.
When the affinity purification process is adopted to purify and separate the mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 of the amino acid sequence of the target protein, the yield and the purity of the purified target protein can be effectively improved, the operation is simple, and the industrial amplification production is easy to realize.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 shows a gel electrophoresis of a product containing a target protein obtained by the affinity purification process in example 1 of the present application.
FIG. 2 shows a gel electrophoresis of the eluted sample from SP Sepharose Fast Flow cation chromatography in example 2 of the present application.
FIG. 3 shows a gel electrophoresis image of an eluted sample obtained by chromatography of phosphocellulose in example 3 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The following is a detailed description of an affinity chromatography packing, a preparation method thereof, and an affinity purification process provided in the embodiments of the present application.
The application provides an affinity chromatography filler, which comprises an activated carrier and a ligand connected to the activated carrier; the structural formula of the ligand is as follows:
Figure BDA0003717917650000041
wherein B is a natural or modified amino-containing nucleobase.
In the ligand structural formula, B may be a natural amino group-containing nucleobase, for example, adenine, guanine, cytosine, or the like; b in the above ligand structural formula may be a modified amino group-containing nucleobase, for example, amino-substituted uracil, amino-substituted adenine, amino-substituted guanine, and amino-substituted cytosine.
The 2 '-O-and 4' -C-positions on the ribose in the ligand structure are bridged by methylene groups to form a 'locked nucleic acid' structure.
It is understood that any substance satisfying the definition of the structural formula described above is included as an alternative ligand in the present application.
The affinity chromatography packing is prepared by connecting the ligand containing a locked nucleic acid structure and an amino group to the activated carrier, so that the mutant T7RNA polymerase containing an amino acid sequence shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7 of the target protein can be effectively separated and purified, and the yield and the purity of the purified target protein are effectively improved.
In this embodiment, the ligand may be selected from commercially available LNA-ATP, LNA-GTP, or LNA-UTP, which has a locked nucleic acid structure and an amino group, and can perform a bioaffinity with a mutated T7RNA polymerase shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, or SEQ ID No.7 as the amino acid sequence of the target protein, so as to achieve specific binding between the ligand and the target protein, thereby purifying the target protein during the affinity purification process.
In this embodiment, the carrier may be selected from agarose gel, polyacrylamide gel, sephadex, porous silica gel or cellulose, which has the properties of hydrophilicity, porous structure, non-specific adsorption property, large specific surface area, high mechanical strength, etc., and is suitable for use as a solid phase carrier in an affinity chromatography column; in addition, the substance contains an activatable reactive group, which is beneficial to the coupling reaction between the activated carrier and the ligand with amino group, thereby immobilizing the ligand on the carrier.
Further, in some embodiments herein, the support may be selected from the group consisting of commercially available CNBr-activated Sepharose 4 Fast Flow (cyanogen bromide activated Sepharose 4FF), which can efficiently perform a coupling reaction with a ligand having an amino group.
The application also provides a preparation method of the affinity chromatography filler, which comprises the steps of carrying out coupling reaction on a ligand and an activated carrier; wherein, the structural formula of the ligand is as follows:
Figure BDA0003717917650000051
wherein B is a natural or modified amino-containing nucleobase.
After being activated, the carrier has an activating group which can carry out coupling reaction with the ligand, thereby being capable of fixing the ligand on the carrier.
In this example, the preparation method of the activated support includes activating the support with an activating agent. The activator may be any one selected from cyanogen bromide, epoxy-based materials, and glutaraldehyde. Further, the epoxy-based material is selected from epichlorohydrin. The substances not only can effectively activate the carrier to fix the ligand on the carrier; and a 'spacing arm' can be formed between the activated carrier and the ligand to increase the distance between the ligand and the carrier, overcome the influence of steric hindrance of the carrier and facilitate the better combination of the ligand and the target protein.
It should be noted that, in other embodiments of the present application, the affinity chromatography packing material may be prepared by a method that does not include activating with an activating agent, and the activated carrier may be used to directly perform a coupling reaction with the ligand, for example, by using commercially available CNBr-activated Sepharose 4 Fast Flow.
When CNBr-activated Sepharose 4 Fast Flow is selected, before the coupling reaction, the CNBr-activated Sepharose 4 Fast Flow needs to be pretreated, which comprises the following steps: washing is carried out at a pH of 2 to 3. Specifically, the pretreatment comprises the steps of suspending and swelling CNBr-activated Sepharose 4 Fast Flow freeze-dried powder in an acidic solution with the pH of 2-3, and then cleaning the solution with the acidic solution.
Illustratively, the mass concentration of CNBr-activated Sepharose 4 Fast Flow freeze-dried powder in an acidic solution is 0.1-0.3g/mL, the acidic solution can be selected from hydrochloric acid, and the washing time can be 10-30 min; further, the washing time may be 15 min. The pretreatment step described above facilitates coupling of the subsequently activated support to the ligand.
In this embodiment, a coupling buffer is used to dissolve the ligand before the coupling reaction between the ligand and the activated carrier. The coupling buffer comprises an inorganic salt buffer which may be selected from carbonate or bicarbonate, such as sodium carbonate or bicarbonate and the like. The concentration of the inorganic salt buffer in the coupling buffer solution is 0.02-0.2M, so that the ligand can be quickly dissolved, the pH value in the coupling buffer solution is stable, and the coupling of the ligand and the activated carrier is facilitated. Illustratively, the concentration of inorganic salt buffer in the coupling buffer may be 0.02M, 0.05M, 0.1M, or 0.2M, among others. Further, the concentration of inorganic salt buffer in the coupling buffer was 0.1M.
The coupling buffer solution also comprises an ionic strength stabilizer which can be selected from sodium chloride; the concentration of the ionic strength stabilizer in the coupling buffer solution is 0.1-1M, which is beneficial to stabilizing the ionic strength in the coupling buffer solution. Illustratively, the concentration of the ionic strength stabilizer in the coupling buffer may be 0.1M, 0.5M, or 1M, among others. Further, the concentration of the ionic strength stabilizer in the coupling buffer was 0.5M.
In this example, the pH of the coupling buffer is 8.0-9.0; further, the pH of the coupling buffer was 8.3.
In this embodiment, the pH of the coupling reaction between the ligand and the activated carrier is 8.0 to 9.0, which is beneficial to promote the coupling between the ligand and the activated carrier. Illustratively, the pH of the coupling reaction of the ligand to the activated support may be 8.0, 8.2, 8.5, 8.8, or 9.0, among others. Further, in some embodiments of the present application, the pH of the coupling reaction of the ligand and the activated carrier is 8.3, which may result in a high coupling efficiency of the ligand and the activated carrier.
It should be noted that, in other embodiments of the present application, the coupling reaction between the ligand and the activated carrier may also be directly performed in a coupling buffer solution in which the ligand is dissolved; after the ligand is dissolved in the coupling buffer solution, the activated carrier to be reacted is added for coupling reaction.
In the embodiment, the temperature for coupling reaction between the ligand and the activated carrier is 4-8 ℃, and the time for coupling reaction is 12-16 h; the above temperature and time may facilitate the coupling reaction. Illustratively, the temperature of the coupling reaction may be 4 ℃, 6 ℃, or 8 ℃, and the like, and the time of the coupling reaction may be 12h, 14h, or 16h, and the like. Further, in some embodiments of the present application, the temperature of the coupling reaction is 4 ℃ and the time of the coupling reaction is 12h, and the coupling effect is better at the above temperature and time.
In some embodiments of the present application, after the coupling reaction, the excess ligand not coupled to the carrier needs to be removed by preliminary washing, and the above-mentioned coupling buffer solution may be selected to remove the excess ligand by preliminary washing.
In some embodiments of the present application, after the removing of the excess ligand by the primary washing, the method further includes removing the residual unconjugated ligand on the carrier by a secondary washing, which may be performed by using a washing solution and a coupling buffer solution alternately, so as to wash away the residual free ligand and prevent the free ligand from being bound to the immobilized ligand. Wherein the cleaning solution comprises sodium acetate and sodium chloride, the concentration of the sodium acetate in the cleaning solution is 0.05-0.3M, the concentration of the sodium chloride in the cleaning solution is 0.2-0.7M, and the pH value of the cleaning solution is 3.5-4.5; further, the concentration of sodium acetate in the cleaning solution is 0.1M, the concentration of sodium chloride in the cleaning solution is 0.5M, and the pH of the cleaning solution is 4.0; the cleaning solution is beneficial to better cleaning the ligand remained on the carrier.
In some embodiments of the present application, the coupling reaction is followed by inactivation of the activated groups in the carrier that are not coupled to the ligand using an inactivation treatment solution. The inactivation treatment solution comprises at least one of a Tris acid primary amine-containing reagent ethanolamine and glycine so as to realize the end group blocking. The concentration of the Tris acid primary amine-containing reagent ethanolamine and glycine in the inactivation treatment liquid is 0.05-0.2M.
The pH value of the inactivation treatment liquid is 7.5-8.5, the inactivation treatment temperature is 10-25 ℃, and the inactivation treatment time is 1-4 h; further, the pH value of the inactivation treatment liquid is 8.0, the inactivation treatment temperature is 20 ℃, and the fire extinguishing treatment time is 2 hours, so that the inactivation effect on the activated groups which are not coupled with the ligand in the carrier can be improved.
The preparation method of the affinity chromatography packing provided by the application at least has the following advantages:
when the affinity chromatography column prepared by the affinity chromatography filler provided by the application is used for purifying and separating the mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 of the amino acid sequence of the target protein, the yield and the purity of the purified target protein can be effectively improved.
The application also provides an affinity purification process, which comprises the steps of adopting the affinity chromatography column provided with the affinity chromatography filler to purify a system containing target protein; the target protein comprises mutant T7RNA polymerase, and the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7.
When the affinity purification process is adopted to purify and separate the mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 of the amino acid sequence of the target protein, the yield and the purity of the purified target protein can be effectively improved, the operation is simple, and the industrial amplification production is easy to realize.
In this example, the purification process involves loading the binding equilibration buffer containing the protein of interest system onto an affinity column, washing with a elution wash buffer, and then eluting with a gradient elution buffer.
Loading a binding equilibrium buffer solution of a mutant T7RNA polymerase containing a target protein amino acid sequence shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7 onto an affinity chromatography column, and specifically binding the target protein with a ligand on the affinity chromatography column to fix the target protein on the affinity chromatography column; eluting and washing the impurities or the non-target proteins which are not combined with the affinity chromatography column and remain on the affinity chromatography column by adopting an eluting and washing buffer solution; and then eluting the affinity chromatographic column combined with the target protein by adopting a gradient elution buffer solution, reducing the affinity action between the target protein and a ligand, desorbing the affinity chromatographic column by using the target protein, and collecting the separated and purified target protein.
In this embodiment, the binding equilibrium buffer includes a first buffer, a first chelating agent, a first reducing agent, and a first stabilizer.
The first buffer is selected from biological buffers or phosphate buffers, for example, biological buffers may be selected from Tris (Tris hydroxymethyl aminomethane), HEPES (4-hydroxyethylpiperazineethanesulfonic acid), MOPS (3- (N-morpholino) propanesulfonic acid), PIPES (piperazine-1, 4-diethylsulfonic acid), BICINE (N, N-dihydroxyethylglycine), CAPS (3- (cyclohexylamine) -1-propanesulfonic acid), TAPS (N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid), EPPS (N- (2-hydroxyethyl) piperazine-N' -3-propanesulfonic acid), MOPSO (3- (N-morpholino) -2-hydroxypropanesulfonic acid), and the like; the phosphate buffer may be potassium dihydrogen phosphate, sodium dihydrogen phosphate, etc. The above substances can effectively maintain the pH required for loading the target protein.
The final concentration of the first buffer in the binding equilibration buffer is 10-100mM, and illustratively, the final concentration of the first buffer in the binding equilibration buffer may be 10mM, 20mM, 30mM, 50mM, or 100mM, and so forth.
Further, in this embodiment, the first buffer is Tris, and the final concentration of Tris in the binding equilibrium buffer is 50 mM.
The first chelating agent is at least one selected from ethylenediamine tetraacetic acid (EDTA), Nitrilotriethanol (NTA), diethylenetriamine pentaacetic acid (DTPA), a substituted salt of ethylenediamine tetraacetic acid and a substituted salt of diethylenetriamine pentaacetic acid, and can effectively prevent the target product from being inactivated by reaction with metal ions in the binding equilibrium buffer. The final concentration of the first chelating agent in the binding equilibrium buffer is 1-10mM, so that the target product can be further prevented from reacting with the metal ions in the binding equilibrium buffer; illustratively, the final concentration of the first chelator in the binding equilibrium buffer may be 1mM, 2mM, 5mM, or 10mM, among others.
Further, in this embodiment, the first chelating agent is EDTA, and the final concentration of EDTA in the binding equilibrium buffer is 5 mM.
The first reducing agent is at least one selected from DTT (dithiothreitol), mercaptoethanol and tris (2-formylethyl) phosphine hydrochloride, and can stabilize the activity of the target protein. The final concentration of the first reducing agent in the binding equilibrium buffer is 1-10mM, so that the activity of the target protein can be further stabilized; illustratively, the concentration of the first reducing agent in the binding equilibration buffer may be 1mM, 5mM, or 10mM, among others.
Further, in this example, the first reducing agent is selected from DTT at a final concentration of 5mM in the binding equilibration buffer.
The first stabilizer is selected from glycerol, and can reduce the polarity of the system to stabilize the activity of the target protein. The final volume concentration of the first stabilizer in the binding equilibrium buffer is 5-15%, if the final concentration of the first stabilizer in the binding equilibrium buffer is low, the purity of the obtained target protein is reduced, and if the final concentration of the first stabilizer in the binding equilibrium buffer is high, the yield of the obtained target protein is reduced; illustratively, the final volume concentration of the first stabilizer in the binding equilibration buffer may be 5%, 10%, or 15%, among others.
Further, in this embodiment, the first stabilizing agent is selected from glycerol, which has a final volume concentration of 10% in the binding equilibration buffer.
In some embodiments, the binding equilibration buffer further comprises an ionic strength stabilizer selected from sodium or potassium salts, for example, the ionic strength stabilizer may be selected from sodium chloride or potassium chloride. The final concentration of the ionic strength stabilizer in the binding equilibration buffer is 10-100 mM.
Further, in this example, the ionic strength stabilizer is selected from sodium chloride, which is present in the binding equilibrium buffer at a final concentration of 50 mM.
In this embodiment, the pH of the binding equilibrium buffer is 7.0-8.0, which facilitates the specific binding of the target protein to the ligand on the affinity chromatography column; illustratively, the pH of the binding equilibration buffer may be 7.0, 7.5, or 8.0, among others. Further, the pH of the binding equilibration buffer was 7.5.
In this embodiment, the elution wash buffer includes a second buffer, a second chelating agent, and a second reducing agent.
The second buffer is selected from phosphate buffers, such as dipotassium phosphate or disodium phosphate, effective to maintain the pH required for elution to remove impurities. The final concentration of the second buffer in the elution wash buffer is 10-100mM, and illustratively, the final concentration of the second buffer in the elution wash buffer can be 10mM, 20mM, 50mM, or 100mM, among others.
Further, in this example, the second buffer is selected from potassium dihydrogen phosphate, which is present in the elution wash buffer at a final concentration of 45 mM.
The second chelating agent is at least one selected from ethylenediamine tetraacetic acid (EDTA), Nitrilotriethanol (NTA), diethylenetriamine pentaacetic acid (DTPA), a substituted salt of ethylenediamine tetraacetic acid and a substituted salt of diethylenetriamine pentaacetic acid, and can effectively prevent the target product from being inactivated by reaction with metal ions in the rinsing impurity buffer solution. The final concentration of the second chelating agent in the washing and impurity-washing buffer solution is 0.5-10mM, so that the reaction of the target product and metal ions in the washing and impurity-washing buffer solution can be further avoided; illustratively, the final concentration of the second chelating agent in the elution wash buffer may be 0.5mM, 1mM, 2mM, 5mM, or 10mM, among others.
Further, in this example, the second chelating agent is selected from EDTA, which is present at a final concentration of 1mM in the elution wash buffer.
The second reducing agent is at least one selected from DTT (dithiothreitol), mercaptoethanol and tris (2-formylethyl) phosphine hydrochloride, and can stabilize the activity of the target protein. The final concentration of the second reducing agent in the leaching and washing mixed buffer solution is 1-10mM, so that the activity of the target protein can be further stabilized; illustratively, the concentration of the second reducing agent in the wash buffer may be 1mM, 5mM, or 10mM, among others.
Further, in this example, the second reducing agent is selected from DTT, which has a final concentration of 5mM in the elution wash buffer.
In some embodiments, the elution wash buffer further comprises an ionic strength stabilizer selected from a sodium or potassium salt, for example, the ionic strength stabilizer may be selected from sodium chloride or potassium chloride. The final concentration of the ionic strength stabilizer in the elution wash buffer was 200-400 mM. Further, in this example, the ionic strength stabilizer is selected from sodium chloride, which is present in the elution buffer at a final concentration of 300 mM.
In this embodiment, the pH of the elution impurity-washing buffer solution is 6.0 to 6.5, which is beneficial to elution removal of impurities or non-target proteins remaining on the affinity chromatography column and not bound to the affinity chromatography column; illustratively, the pH of the elution wash buffer can be 6.0, 6.2, or 6.5, among others. Further, the pH of the elution wash buffer was 6.4.
In this embodiment, the gradient elution buffer includes a third buffer, a third chelating agent, a third reducing agent, and a third stabilizing agent.
The third buffer is selected from acetate buffers, such as sodium acetate; the pH required for gradient elution can be effectively maintained. The final concentration of the third buffer in the gradient elution buffer is 10-100mM, and illustratively, the final concentration of the third buffer in the gradient elution buffer may be 10mM, 20mM, 50mM, or 100mM, and so forth. Further, in this example, the third buffer is selected from sodium acetate, which is present in the gradient elution buffer at a final concentration of 50 mM.
The third chelating agent is at least one selected from ethylenediamine tetraacetic acid (EDTA), Nitrilotriethanol (NTA), diethylenetriamine pentaacetic acid (DTPA), a substituted salt of ethylenediamine tetraacetic acid and a substituted salt of diethylenetriamine pentaacetic acid, and can effectively prevent the target product from being inactivated by reaction with metal ions in the gradient elution buffer. The final concentration of the third chelating agent in the gradient elution buffer solution is 0.5-10mM, so that the target product can be further prevented from reacting with metal ions in the gradient elution buffer solution; illustratively, the final concentration of the third chelating agent in the gradient elution buffer may be 0.5mM, 1mM, 2mM, 5mM, or 10mM, among others. Further, in this example, the third chelating agent is selected from EDTA, which is present at a final concentration of 1mM in the gradient elution buffer.
The third reducing agent is at least one selected from DTT (dithiothreitol), mercaptoethanol and tris (2-formylethyl) phosphine hydrochloride, and can stabilize the activity of the target protein. The final concentration of the third reducing agent in the gradient elution buffer solution is 5-20mM, so that the activity of the target protein can be further stabilized; illustratively, the concentration of the third reducing agent in the gradient elution buffer may be 5mM, 10mM, or 20mM, among others. Further, in this example, the third reducing agent is selected from DTT, which is present in the gradient elution buffer at a final concentration of 10 mM.
The third stabilizer, glycerol alone, can reduce the polarity of the system to stabilize the activity of the target protein. The final volume concentration of the third stabilizer in the gradient elution buffer solution is 5-15%, so that the polarity of the system can be further reduced; illustratively, the final volume concentration of the third stabilizing agent in the gradient elution buffer may be 5%, 10%, or 15%, and so forth. Further, in this example, the third stabilizing agent is selected from glycerol, which has a final volume concentration of 10% in the gradient elution buffer.
In some embodiments, the gradient elution buffer further comprises an ionic strength stabilizer selected from a sodium or potassium salt, for example, the ionic strength stabilizer may be selected from sodium chloride or potassium chloride. The final concentration of the ionic strength stabilizer in the gradient elution buffer solution is 50mM-4M, which is beneficial to better separating the target protein from the ligand so as to obtain the required target protein; further, in this example, the ionic strength stabilizer was selected from sodium chloride, which was present in the gradient elution buffer at a final concentration of 3M.
In this embodiment, the gradient elution buffer has a pH of 4.5 to 5.5, which can facilitate the separation of the target protein and the ligand, thereby obtaining the desired target protein; illustratively, the pH of the gradient elution buffer may be 4.5, 5.0, or 5.5, among others. Further, the pH of the gradient elution buffer was 5.0.
In some embodiments of the present application, the affinity chromatography column is further washed and regenerated with high and low pH regeneration buffers alternately after elution, so that the affinity chromatography column can be reused for multiple times.
In some embodiments, the high pH regeneration buffer has a pH of 7-10, further the high pH regeneration buffer has a pH of 8.5, and the high pH regeneration buffer comprises Tris buffer. The low pH regeneration buffer has a pH of 4-5, further the low pH regeneration buffer has a pH of 4.5, and the low pH regeneration buffer comprises sodium acetate inorganic salt buffer. The high and low pH regeneration buffer solutions both contain sodium chloride, and the concentrations of the sodium chloride in the high and low pH regeneration buffer solutions are both 0.5M.
Further, in some embodiments of the present application, the regenerating treatment further comprises storing the regenerated affinity chromatography column in a long-term storage solution for later use. The long-term preservation solution contains 0.1 percent of sodium azide by mass fraction, and the preservation temperature is 2-8 ℃.
In order to further improve the purification effect of the mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7, the target protein is further purified by cation chromatography and phosphocellulose chromatography after the target protein is purified by the affinity purification process provided by the present application.
The characteristics and properties of an affinity chromatography packing, a preparation method thereof, and an affinity purification process provided by the present application are further described in detail with reference to the following examples.
Example 1
The embodiment provides an affinity chromatography filler, a preparation method thereof and an affinity purification process, and specifically comprises the following steps:
(1) preparation of affinity chromatography packing:
60g of CNBr-activated Sepharose 4 Fast Flow freeze-dried powder is suspended and swelled in 450mL of 1mM hydrochloric acid solution with pH 3 for 30min, and then 6759mL of 1mM hydrochloric acid solution is used for washing after being resuspended by a glass filter, so that the activated carrier to be subjected to coupling reaction is obtained.
1.2g of LNA-ATP freeze-dried powder is dissolved in 100mL of coupling buffer solution with the pH value of 8.3 to obtain a system to be coupled. Wherein the coupling buffer solution is prepared from NaHCO 3 And NaCl in ultrapure water, coupling with NaHCO in buffer 3 The concentration of (2) was 0.1M, and the concentration of NaCl was 0.5M.
And adding the activated carrier to be coupled into 225mL of a system to be coupled, slightly stirring uniformly, and incubating for 12h at 4 ℃ under mild stirring to perform coupling reaction. And (3) cleaning the coupled system by adopting 1500mL of the coupling buffer solution, cleaning for 2h at 20 ℃ by adopting 1500mL of inactivation treatment solution with the pH of 8.0, and then alternately cleaning by adopting 3000mL of cleaning solution with the pH of 4.0 and 1500mL of the coupling buffer solution to prepare the affinity chromatography filler. The inactivation treatment solution is prepared by dissolving Tris-HCl in ultrapure water, and the concentration of the Tris-HCl in the inactivation treatment solution is 0.1M; the cleaning solution is prepared by dissolving NaAC and NaCl in ultrapure water, wherein the concentration of NaAC in the cleaning solution is 0.1M, and the concentration of NaCl in the cleaning solution is 0.5M.
(2) Preparation of target protein:
aiming at the T7RNA polymerase with the mutation of which the amino acid sequence is shown as SEQ ID NO.1, the gene sequence of the T7RNA polymerase with the correct mutation sequence is amplified through a polymerase chain reaction, and the amplified gene sequence is transferred into escherichia coli. Firstly, culturing the escherichia coli in an LB culture medium for 12 hours,then cultured in 2xYT medium at 37 ℃ and 180rpm to OD 600 The value is 0.3, then the culture system is cooled to 20 ℃ and slowly expanded to OD 600 The value was 0.5 and expression was induced for 12h by addition of IPTG to a final concentration of 0.2 mM. And centrifuging to obtain the precipitate to obtain the thallus.
The harvested thalli after expression is subjected to membrane rupture twice by a homogenizer at 650psi, and centrifuged for 30min at the rotating speed of 8500rpm of a desktop centrifuge and the temperature of 4 ℃, and then supernatant is harvested. Slowly adding ammonium sulfate solution into the supernatant during the mild stirring process, stirring at 4 ℃ for 2h, centrifuging at 8500rpm for 50min to obtain precipitate, and finally resuspending with a dimension buffer and dialyzing by 300 times. Collecting the supernatant to obtain the system containing the target protein.
Wherein, the LB culture medium contains Amresco soy peptide 10g, Merck Yeast Extract5g, NaCl 10g kanamycin to the final concentration of 60 ug/mL; 2xYT culture medium contains 16g of Amresco soy Peptone, 5g of Merck Yeast Extract and 5g of NaCl containing 60ug/mL of kanamycin; adding the ammonium sulfate solution into a sample volume which is 0.85 times of the volume of the ammonium sulfate solution, wherein the ammonium sulfate solution is saturated ammonium sulfate solution at the temperature of 0 ℃; the dimension buffer contains 20mM Tris, 1mM EDTA, 5mM DTT, 5% glycerol, 20ug/mL PMSF, 2.5ug/mL Leuppeptin pH7.5.
(3) An affinity purification process:
diluting the system containing the target protein obtained in the step (2) by using a binding equilibrium buffer solution with the pH of 7.5 until the pH fluctuation of the system is within +/-0.2 and the conductivity fluctuation of the system is within +/-5 ms/cm, and loading the system to an affinity chromatography column provided with the affinity chromatography filler obtained in the step (1). And then washing the mixed buffer solution by 10 column volumes by adopting a leaching and washing buffer solution with the pH value of 6.4, then eluting by 10 column volumes by adopting a gradient elution buffer solution with the pH value of 5.0, and collecting the product containing the target protein.
The binding equilibrium buffer solution is prepared by mixing and dissolving Tris, EDTA, NaCl, DTT and 99.7 mass percent of glycerol in ultrapure water, wherein the concentrations of Tris, EDTA, NaCl, DTT and glycerol in the binding equilibrium buffer solution are 50mM, 5mM, 50mM, 5mM and 10 percent respectively. The leaching impurity-washing buffer solution is prepared by mixing and dissolving monopotassium phosphate, EDTA, NaCl and DTT in ultrapure water, wherein the concentrations of the monopotassium phosphate, the EDTA, the NaCl and the DTT in the leaching impurity-washing buffer solution are 45mM, 1mM, 300mM and 5mM respectively. The gradient elution buffer is prepared by mixing NaAC, EDTA, DTT, NaCl and 99.7% glycerol by mass fraction and dissolving in ultrapure water, wherein the concentrations of NaAC, EDTA, DTT, NaCl and glycerol in the gradient elution buffer are respectively 50mM, 1mM, 10mM, 3M and 10% (volume concentration).
Example 2
Example 2 differs from example 1 in that: after the step (3), the products containing the target protein obtained after elution in the step (3) are sequentially purified by SP Sepharose Fast Flow cationic chromatography.
The SP Sepharose Fast Flow cation chromatography purification step comprises: dialyzing the product containing the target protein obtained in the step (3) in a cation chromatography equilibrium buffer solution with the pH value of 7.51 until the pH value of the system fluctuates within +/-0.2 and the electric conductivity value of the system fluctuates within +/-5 ms/cm. Then, the sample was loaded, and then eluted with a cation chromatography elution buffer having a pH of 7.52, and the eluted sample was collected.
The cation chromatography equilibrium buffer solution is prepared by mixing and dissolving Tris, EDTA, NaCl, DTT and 99.7 mass percent of glycerol in ultrapure water, wherein the concentrations of the Tris, EDTA, NaCl, DTT and glycerol in the cation chromatography equilibrium buffer solution are respectively 50mM, 5mM, 10mM, 5mM and 10%. The cation chromatography elution buffer solution is prepared by mixing and dissolving Tris, EDTA, NaCl, DTT and 99.7 mass percent of glycerol in ultrapure water, wherein the concentrations of Tris, EDTA, NaCl, DTT and glycerol in the cation chromatography elution buffer solution are respectively 50mM, 5mM, 1M, 5mM and 10 percent.
Example 3
Example 3 differs from example 1 in that: and (3) after the step (3), sequentially carrying out phosphocellulose chromatography purification on the product containing the target protein obtained after elution in the step (3).
The chromatographic purification step of the phosphocellulose comprises the following steps: dialyzing an elution sample obtained by cation chromatography elution in a cellulose chromatography equilibrium buffer solution with the pH value of 6.5 until the pH fluctuation of a system is within +/-0.2 and the electric conductivity value fluctuation of the system is within +/-5 ms/cm; then, the sample was loaded, and then eluted with 10 column volumes using a cellulose elution buffer at pH 6.5, and the eluted sample was collected.
Wherein the cellulose phosphate chromatography equilibrium buffer solution is prepared by mixing and dissolving monopotassium phosphate, EDTA, DTT and 99.7% glycerol in ultrapure water, and the concentrations of the monopotassium phosphate, the EDTA, the DTT and the glycerol in the cellulose chromatography equilibrium buffer solution are respectively 10mM, 0.1mM, 1mM and 5%. The cellulose phosphate chromatography elution buffer solution is prepared by mixing and dissolving Potassium dihydrogen phosphate, EDTA, DTT and 99.7% glycerol in ultrapure water, wherein the concentrations of the Potassium dihydrogen phosphate, the EDTA, the DTT and the glycerol in the cellulose chromatography equilibrium buffer solution are respectively 500mM, 0.1mM, 1mM and 5%.
Example 4
Example 4 differs from example 1 in that: the pH of the coupling buffer was 9.0.
Example 5
Example 5 differs from example 1 in that: the pH of the coupling buffer was 8.0.
Example 6
Example 6 differs from example 1 in that: the glycerol concentration in the binding equilibration buffer was 5% by volume.
Example 7
Example 7 differs from example 1 in that: the glycerol concentration in the binding equilibration buffer was 15% by volume.
Comparative example 1
Comparative example 1 differs from example 1 in that: comparative example 1 the target protein is native T7RNA polymerase.
Comparative example 2
Comparative example 2 differs from example 1 in that: and (3) difference.
Step (3) of comparative example 2 was: sequentially carrying out High S cation chromatography, Blue column chromatography, anion Q column chromatography and hydroxyapatite filler chromatography.
High S cation chromatography: loading a system containing the target protein into a cation chromatographic column, and enabling a cation chromatographic layer to flow through in an equilibrium flow-through liquid with the pH value of 7.7; wherein, the equilibrium flow-through liquid contains 20mM sodium dihydrogen phosphate, 1mM EDTA, 5mM DTT, 5% glycerol and 2.5 mug/mL Leuteptin (protease inhibitor) in mass fraction, and the flow-through sample is collected.
Blue column chromatography: dialyzing the flow-through sample collected by High S cation chromatography by using dialysis balance liquid with pH of 8.0 for 2-3 times, wherein the total dialysis liquid change is 100 times, and the dialysis time is 4h each time; and (3) loading the sample obtained after dialysis to a Blue column, washing by using a washing buffer solution with the pH of 8.0, eluting by using a gradient eluent with the pH of 8.0, and collecting an eluted sample. Wherein the dialysis balance solution has KH concentration of 10mM 2 PO 4 200mM NaCl, glycerol with the mass fraction of 5%, 1mM DTT, 1mM EDTA, 20 mu g/mL PMSF (phenylmethylsulfonyl fluoride) and 2.5 mu g/mL Leuteptin; the elution buffer had a KH concentration of 10mM 2 PO 4 230mM NaCl, glycerol with the mass fraction of 5%, 1mM DTT and 1mM EDTA; gradient eluent has KH concentration of 10mM 2 PO 4 1.5M NaCl, glycerol with the mass fraction of 5%, 1mM DTT and 1mM EDTA.
Anion Q column chromatography: dialyzing the elution sample collected by Blue column chromatography by using dialysate with pH of 7.9 for 2-3 times, wherein the total dialysis time is 4 h; and loading the sample obtained after dialysis to an anion Q column, eluting by using elution buffer with the pH of 7.9, eluting by using gradient eluent with the pH of 7.9, and collecting an eluted sample. Wherein the dialysate comprises 15mM Tris, 10mM NaCl, 1mM DTT, 1mM EDTA, and 0.5% Triton X-100 (polyethylene glycol octyl phenyl ether); the elution buffer comprises 20mM Tris, 0.5% Triton X-100, 5% glycerol, 1mM DTT, 1mM EDTA and 30mM NaCl; the gradient eluent comprises 20mM Tris, 0.5% Triton X-100, 5% glycerol, 1mM DTT, 1mM EDTA and 200mM NaCl.
Chromatography of hydroxyapatite filler: dialyzing the elution sample collected by the anion Q column chromatography by using dialysate with pH of 8.0 for 2-3 times, wherein the total dialysis time is 100 times, and each time of dialysis is 4 hours; subjecting the dialyzed sampleAnd (4) loading the sample to hydroxyapatite filler, eluting by adopting gradient eluent with the pH value of 8.0, and collecting an eluted sample. Wherein the dialysate has KH concentration of 10mM 2 PO 4 1mM DTT, 0.1mM EDTA, 5% glycerol, 0.5% NP-40 (ethylphenylpolyethylene glycol), 0.5% Tween 20 (polyoxyethylene sorbitan monolaurate); the gradient eluent has a KH concentration of 500mM 2 PO 4 1mM DTT, 0.1mM EDTA, 5% glycerol, 0.5% NP-40, and 0.5% Tween 20.
Test example 1
Gel electrophoresis experiments were performed on the products containing the target protein obtained by the affinity purification process in step (3) of example 1, respectively, and the results are shown in fig. 1. Gel electrophoresis experiments were performed on the elution sample obtained by the SP Sepharose Fast Flow cation chromatography of example 2 and the elution sample obtained by the phosphocellulose chromatographic purification of example 3, and the results are shown in FIGS. 2 and 3, respectively.
Wherein, in fig. 1, the label "load": the sample before the column is marked with a Marker M, and the sample is collected by affinity chromatography gradient elution with the number of 1-40. In fig. 2, the label "load": as pre-column samples, the numbers 1-14 are all cation chromatography gradient elution collection samples. In FIG. 3, the symbol "M" is Marker, and the numbers 1 to 43 are all phosphocellulose chromatographic gradient elution collected samples.
As can be seen from FIG. 1, the affinity purification process provided in example 1 enables efficient collection of the target protein (i.e., the mutant T7RNA polymerase having the amino acid sequence shown in SEQ ID NO. 1). As can be seen from fig. 2 and 3, after the affinity purification process provided in the present application is performed, the yield of the target protein can be further improved by purifying SP Sepharose Fast Flow cation chromatography or phosphocellulose chromatography.
Test example 2
Statistics were made on the yields and purities of the target proteins obtained by the purification methods provided in examples 1 to 7 and comparative examples 1 to 2, and the results are shown in Table 1.
TABLE 1
Figure BDA0003717917650000211
As can be seen from Table 1, the yields of the target proteins (i.e., the mutated T7RNA polymerase with the amino acid sequence shown in SEQ ID NO. 1) obtained by the purification methods provided in examples 1-7 are significantly higher than the yield of the purification method provided in comparative example 1, which indicates that the purification process of the present application can achieve specific affinity adsorption for the T7RNA polymerase with a specific amino acid sequence, thereby achieving high-efficiency separation of the target proteins and the impurity proteins.
The yield of the target protein obtained by the purification methods provided in examples 1-7 is significantly higher than that of the purification method provided in comparative example 2, which shows that the purification process of the present application can effectively improve the yield and purity of the target protein obtained after purification compared with the conventional four-step chromatography in comparative example 2, and has the advantages of simple operation, few processes, and greatly reduced input cost of manpower and material resources and time cost.
Examples 2-3 provide purification methods that yield and purity of the target protein slightly higher than the purification method provided in example 1, which shows that after the affinity purification process provided herein, SP Sepharose Fast Flow cation chromatography purification or phosphocellulose chromatography purification can further improve the yield and purity of the target protein.
Examples 4-5 provide purification methods that yield the target protein slightly lower than the purification method provided in example 1, indicating that the pH of the coupled ligand and the activated support during the preparation of the affinity chromatography packing can affect the final target protein yield, because the pH of the coupled ligand and the activated support can affect the coupling ratio between the ligand and the activated support, and thus affect the specific affinity adsorption of the affinity chromatography packing to the target protein. The coupling reaction in example 1 has a pH of 8.3, and the purity and yield of the target protein are both good.
Example 6 provides a purification method to obtain a slightly higher yield of the target protein than example 1, and a slightly lower purity of the target protein than example 1; example 7 provides a purification method to obtain a slightly lower yield of the target protein than example 1, and a slightly higher purity of the target protein than example 1; the experimental results show that the volume concentration of the glycerol in the combined equilibrium buffer solution can influence the yield and the purity of the target protein; the glycerol concentration in the binding equilibrium buffer of example 1 is 10% by volume, and the purity and yield of the target protein are both good.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shanghai megadimensional science and technology development Co., Ltd
SHANGHAI ZHAOWEI BIOENGINEERING Co.,Ltd.
<120> affinity chromatography filler, preparation method and affinity purification process thereof
<130> 2022.06.23
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 883
<212> PRT
<213> Artificial sequence
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Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
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Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
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Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
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Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
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Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
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Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
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Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
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Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
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Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
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Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
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His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
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Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
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Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
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Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
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Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
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Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
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Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
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Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
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Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
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Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
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His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
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Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
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Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
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Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
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Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
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Ala Phe Ala
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Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
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Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
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Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
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Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
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Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
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Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
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Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
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Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
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His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
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Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
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Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
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Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
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Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
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Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
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Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
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Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
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Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 3
<211> 883
<212> PRT
<213> Artificial sequence
<400> 3
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 4
<211> 883
<212> PRT
<213> Artificial sequence
<400> 4
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 5
<211> 883
<212> PRT
<213> Artificial sequence
<400> 5
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 6
<211> 883
<212> PRT
<213> Artificial sequence
<400> 6
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 7
<211> 883
<212> PRT
<213> Artificial sequence
<400> 7
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala

Claims (10)

1. An affinity chromatography packing, which is characterized by comprising an activated carrier and a ligand connected to the activated carrier; the structural formula of the ligand is as follows:
Figure FDA0003717917640000011
wherein B is a natural or modified amino-containing nucleobase.
2. The affinity chromatography packing according to claim 1, wherein the ligand is selected from LNA-ATP, LNA-GTP or LNA-UTP;
optionally, the carrier is selected from sepharose, polyacrylamide gel, sephadex, porous silica gel or cellulose.
3. The method of claim 1 or 2, comprising coupling the ligand to the activated support.
4. The method of preparing an affinity chromatography packing material of claim 3, wherein the activated support is prepared by activating the support with an activating agent;
the activating agent is any one of cyanogen bromide, epoxy materials and glutaraldehyde;
optionally, the epoxy-based material is selected from epichlorohydrin.
5. The method for preparing the affinity chromatography packing according to claim 3, wherein the pH of the coupling reaction is 8.0 to 9.0;
alternatively, the pH of the coupling reaction is 8.3.
6. The method for preparing the affinity chromatography packing according to claim 3, wherein the temperature of the coupling reaction is 4-8 ℃, and the time of the coupling reaction is 12-16 h;
optionally, the temperature of the coupling reaction is 4 ℃ and the time of the coupling reaction is 12 h.
7. An affinity purification process comprising subjecting a system containing a target protein to a purification treatment using an affinity chromatography column on which the affinity chromatography packing of claim 1 or 2 is placed;
the target protein comprises mutant T7RNA polymerase, and the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7.
8. The affinity purification process according to claim 7, wherein the purification treatment comprises loading a binding equilibrium buffer of the system containing the protein of interest onto the affinity chromatography column, washing with a elution wash buffer, and eluting with a gradient elution buffer;
wherein the binding equilibration buffer comprises a first buffer, a first chelating agent, a first reducing agent, and a first stabilizing agent; the rinsing and impurity washing buffer solution comprises a second buffer, a second chelating agent and a second reducing agent; the gradient elution buffer comprises a third buffer, a third chelating agent, a third reducing agent, and a third stabilizing agent;
the first buffer is selected from a biological buffer or a phosphate buffer; the second buffer is selected from phosphate buffers; the third buffer is selected from an acetate buffer; the first chelating agent, the second chelating agent, and the third chelating agent are each independently at least one selected from the group consisting of ethylenediaminetetraacetic acid, nitrilotriethanol, diethylenetriaminepentaacetic acid, substituted salts of ethylenediaminetetraacetic acid, and substituted salts of diethylenetriaminepentaacetic acid; the first reducing agent, the second reducing agent and the third reducing agent are respectively and independently selected from at least one of DTT, mercaptoethanol and tris (2-formylethyl) phosphine hydrochloride; the first stabilizer and the third stabilizer are each independently selected from glycerol.
9. The affinity purification process of claim 8, wherein the final concentrations of the first buffer, the first chelator, the first reducing agent, and the first stabilizer in the binding equilibration buffer are 10-100mM, 1-10mM, and 5-15%, respectively;
the final concentrations of the second buffer, the second chelating agent and the second reducing agent in the rinsing and impurity-washing buffer are respectively 10-100mM, 0.5-10mM and 1-10 mM;
the final concentrations of the third buffer, the third chelating agent, the third reducing agent, and the third stabilizing agent in the gradient elution buffer are 10-100mM, 0.5-10mM, 5-20mM, and 5-15%, respectively.
10. The affinity purification process of claim 8, wherein the binding equilibration buffer has a pH of 7.0-8.0; the pH value of the leaching impurity-washing buffer solution is 6.0-6.5; the pH value of the gradient elution buffer solution is 4.5-5.5;
optionally, the binding equilibration buffer has a pH of 7.5; the pH value of the leaching impurity-washing buffer solution is 6.4; the gradient elution buffer had a pH of 5.0.
CN202210749834.7A 2022-06-28 2022-06-28 Affinity chromatography filler, preparation method thereof and affinity purification process Pending CN114939400A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012087231A1 (en) * 2010-12-20 2012-06-28 Ge Healthcare Bio-Sciences Ab Affinity chromatography matrix
CN106140099A (en) * 2015-04-17 2016-11-23 北京美正生物科技有限公司 A kind of immune affinity column of isolated and purified lactoferrin and its production and use
CN113416243A (en) * 2021-08-23 2021-09-21 上海盛迪医药有限公司 Method for purifying antibody

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012087231A1 (en) * 2010-12-20 2012-06-28 Ge Healthcare Bio-Sciences Ab Affinity chromatography matrix
CN106140099A (en) * 2015-04-17 2016-11-23 北京美正生物科技有限公司 A kind of immune affinity column of isolated and purified lactoferrin and its production and use
CN113416243A (en) * 2021-08-23 2021-09-21 上海盛迪医药有限公司 Method for purifying antibody

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