WO2014146350A1 - 一类突变的具有高耐碱特性的蛋白a及其应用 - Google Patents

一类突变的具有高耐碱特性的蛋白a及其应用 Download PDF

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Publication number
WO2014146350A1
WO2014146350A1 PCT/CN2013/076445 CN2013076445W WO2014146350A1 WO 2014146350 A1 WO2014146350 A1 WO 2014146350A1 CN 2013076445 W CN2013076445 W CN 2013076445W WO 2014146350 A1 WO2014146350 A1 WO 2014146350A1
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protein
immunoglobulin
seq
multimer
primer
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PCT/CN2013/076445
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English (en)
French (fr)
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钱红
白涛
花榕
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南京金斯瑞生物科技有限公司
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Priority to US14/778,395 priority Critical patent/US10287327B2/en
Publication of WO2014146350A1 publication Critical patent/WO2014146350A1/zh
Priority to US16/169,376 priority patent/US10464972B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion

Definitions

  • Biotechnology is one of the fastest growing areas of high technology in the world today.
  • As an antibody drug in the field of biotechnology in recent years, while continuously improving the health and quality of life of patients, it has also achieved remarkable market performance.
  • innovative drugs are significantly reduced, and many small molecule drugs are currently facing the threat of patent cliffs. Therefore, in order to seek new growth points, many pharmaceutical companies, especially biotechnology pharmaceutical companies, have gradually entered the field of antibody drug research and development.
  • antibody drugs have been widely used in the diagnosis and treatment of basic biomedical research, diseases (such as cancer, organ transplant rejection, autoimmune diseases, etc.).
  • the purified chromatographic medium required for production can be used repeatedly, the production cost of the antibody can be significantly reduced.
  • the chromatographic medium leaves the uneluted protein every time the antibody is purified, the protein aggregates even leave substances harmful to the human body, such as viruses and endotoxins. Therefore, the medium must be cleaned when it is reused.
  • the most effective way to recover the chromatographic medium is to use an alkaline method called in-situ cleaning.
  • the standard procedure includes treating the purified medium with 0. 5M Na0H. The method can effectively remove impurities, but it is likely to damage the purification medium.
  • the alkali-resistant and immunoglobulin-binding protein A molecule of the present invention can be used as a good ligand on an affinity medium for purifying antibody drugs, and the purification medium can also be cleaned by an alkaline method in situ. The purified medium after use is used for regeneration. Summary of the invention
  • the object of the present invention is to provide a class of mutated protein A having high alkali resistance characteristics in view of the above-mentioned deficiencies of the prior art. Another object of the invention is to provide the use of this protein A.
  • a class of genetically mutated protein A having an amino acid sequence as set forth in SEQ ID N0.1 or SEQ ID N0.2, or in combination with an amino acid sequence represented by SEQ ID N0.1 or SEQ ID N0.2 in immunoglobulin
  • the region has 99% or more homology of all amino acid sequences capable of binding immunoglobulin, wherein the immunoglobulin binding region is the 7th to 54th amino acid residues.
  • a nucleic acid encoding the protein A is A nucleic acid encoding the protein A.
  • the protein A involved in the present invention is essentially a kind of molecule which can bind immunoglobulin, and the molecule can firmly bind to other regions outside the complementarity determining region of the immunoglobulin, and can withstand the strong alkaline environment of PH13-14.
  • the protein conformation of the immunoglobulin-binding region of this protein A is mainly composed of three alpha helices and is folded into a three-helix bundle structure. It binds to the Fc region of an immunoglobulin via alpha helix 1 and 2 surface amino acid residues, or binds to a non-complementarity determining region on an immunoglobulin Fab via alpha helix 2 and 3 surface residues.
  • the hydrophobic residues on the surface of the ⁇ -helix 1, 2, 3 form a hydrophobic core at the center of the helical bundle, which can tightly bind the ⁇ -helices 1, 2, and 3 to maintain a stable structure of the protein.
  • the ⁇ -helices 1, 2 , and 3 will stretch and stretch, but strong.
  • the hydrophobic force still allows the alpha helices 1, 2, 3 to tightly bond and ensure that their relative positions do not change significantly.
  • the coupling of the alkali-resistant peptone and its multimer to a solid phase carrier can be used to isolate and purify immunoglobulin molecules or to couple them with a labeling agent for detecting immunoglobulin molecules, and if As a ligand for the chromatographic medium, the chromatographic medium can be regenerated by in-situ alkali cleaning.
  • the protein A monomer of the present invention has a molecular weight of about 6 KD and is composed of 58 amino acids, and can be obtained by chemical synthesis of a polypeptide or cell expression. If cell expression is used, a cell line carrying the gene encoding the amino acid sequence of the protein must first be constructed. Therefore, it is necessary to proceed according to the following procedure: First, the gene encoding the amino acid sequence of protein A is obtained by gene synthesis, and next, the gene encoding the amino acid sequence of protein A is recombined into an expression vector, and the vector can be added and purified as needed. The labeling label or the indicator label, followed by the stable transfer of the expression vector into the appropriate cells, completes the entire expression system that can produce such protein A.
  • the desired protein A (including monomeric or multimeric) having a binding immunoglobulin molecule can be obtained from a culture medium or a cell.
  • Must pay attention to protein A The expression in the cell needs to be tightly controlled. Therefore, it is very important to select the vector for expressing protein A. It should have the following characteristics: 1.
  • the expression vector contains a promoter or an initial transcription site, and 2 the expression vector contains an operon. To switch the expression of the gene, 3.
  • the expression vector contains a ribosome binding site, 4.
  • the expression vector contains transcription and translation termination sites, which can improve the stability of transcription and translation products.
  • the vectors expressing protein A include PET (Novagen), PQE30 (Qiagen), PGS21a (genscript), pGAPZa A (Invitrogen), etc., and the corresponding host cells are selected from Escherichia coli genetically engineered bacteria and Pichia pastoris.
  • Such egg SA can be produced in the cells of the host, extracellularly or on the cell membrane.
  • Such protein A can be isolated and purified by various separation operations using physical properties, chemical properties and the like as needed. Specifically, for example, general protein precipitant treatment (salting method), centrifugation, osmotic crushing, ultrasonication, ultrafiltration, gel filtration, and adsorption chromatography, ion exchange chromatography, affinity chromatography, high-speed liquid phase Various liquid chromatography such as chromatography, dialysis, and combinations of these methods. Furthermore, by expressing a protein in which such a protein is fused to an affinity tag, affinity purification can be performed using the label. Affinity tags as described herein, such as polyhistidine tags and FLAG tags. Purification of such protein A can be carried out using these markers.
  • the activity of such protein A binding immunoglobulin can be determined by an ELISA assay.
  • the protein A is immobilized on a solid phase carrier, and then the enzyme-labeled immunoglobulin is added to adsorb on the protein A, and the complex of the immunoglobulin and the protein A formed on the solid phase carrier is washed and washed.
  • the other substances in the liquid are separated, such that the amount of the complex formed by the protein A and the immunoglobulin on the solid phase carrier is in a one-to-one ratio with the amount of the enzyme.
  • the reaction substrate of the enzyme is added, the substrate is enzymatically converted into a colored product, so that the amount of protein A-bound immunoglobulin can be qualitatively and quantitatively analyzed according to the depth of coloration.
  • Such protein A is first immersed in 0.5 M NaOH for 60 hours, and the activity of binding immunoglobulin is determined according to the ELISA test described above, how can the immunoglobulin activity still be well maintained, then This proves that such protein A is stable in a strong alkaline environment. Further coupling such protein A to a solid phase by, for example, using an amino or carboxyl group present in such protein A, via a coupling agent such as a diepoxide, epichlorohydrin, cyanogen bromide or N-hydroxysuccinamide.
  • a coupling agent such as a diepoxide, epichlorohydrin, cyanogen bromide or N-hydroxysuccinamide.
  • the protein A discussed above is a monomer, however this protein can be combined into a multimeric protein such as a dimer, a trimer, a tetramer or the like.
  • a polymer A belonging to the present invention which is formed by itself or with other proteins A in the present invention, is also another aspect of the present invention.
  • the multimer of the present invention comprises a monomer unit linked by a one-stage amino acid sequence of preferably 4 to 10 amino acids. This linkage does not destroy the spatial conformation of protein A, but is also sufficiently stable in an alkaline environment without compromising the properties of protein A.
  • the multimer is a dimer of protein A, wherein connexin A is 4 amino acids in length (ADGK).
  • the present invention relates to a nucleic acid sequence encoding the above protein A or a multimer thereof.
  • the nucleic acid sequence is codon-optimized, and the optimized nucleic acid sequence avoids the generation of rare codons and the formation of secondary structures, and is synthesized by primer-bridge artificial gene synthesis.
  • the present invention relates to all protein derivatives of such protein A which are chemically modified by N-terminal or C-terminal or side-chain groups and which are capable of binding strongly to immunoglobulin.
  • an N-acetyl polypeptide is obtained by acetylation of an N-terminal amino group, and is acylated with a fatty acid to obtain an N-fatty acid acylated polypeptide.
  • a C-terminal carboxyl group is converted into an amide to obtain a polypeptide amide, which is converted into an ester to obtain a polypeptide ester, which is converted into a thioester to obtain a polypeptide thioester.
  • Derivatization modification plays an important role in regulating the acidity and alkalinity, stability, solubility, reactivity and biological activity of the original protein. If a cysteine residue is provided at the C-terminus of protein A, protein A can be coupled to the carrier via a thioether linkage, and such coupling methods are readily performed by standard techniques and equipment.
  • the present invention relates to commercial applications of such protein A, including purification of immunoglobulins and detection of immunoglobulins.
  • the use of immunoglobulin purification includes a method of layer affinity separation in which at least one target compound is separated from a complex matrix by adsorption to protein A or multimer as described above, which is a widely used separation. technology.
  • the specific step includes flowing the sample solution containing the immunoglobulin through the protein A chromatography medium. In this step, the other components of the solution will pass through substantially unimpeded, while the immunoglobulin is adsorbed onto the chromatographic medium.
  • the medium is then washed, for example with a phosphate buffer, to remove residual non-specifically bound impurities, as discussed above, also by a washing step using an alkaline reagent.
  • the eluent is passed through a chromatographic medium to elute the adsorbed immunoglobulin, which is usually achieved by changing the pH, ionic strength or adding a competitive substance.
  • the use of immunoglobulin detection includes a method for detecting immunoglobulins, including first enzymatic labeling on protein A, chemiluminescent labeling or Isotope labeling, and then adsorption to the immunoglobulin by protein A, can achieve immunoglobulin visual analysis.
  • the protein A and its multimers and derivatives provided by the present invention can be firmly bound to other regions of the immunoglobulin molecule except the complementarity determining region, and can be used as a ligand coupled to a solid phase carrier for isolating immunoglobulin.
  • This protein A maintains chemical stability in a pH 13-14 strong base environment and acts as a chromatographic ligand to withstand harsh in-situ cleaning conditions.
  • the protein A of the invention and its multimers and derivatives can be used in the purification and/or detection of immunoglobulins.
  • Figure 1 SDS-PAGE was used to detect the expression of protein A in E. coli. Lanes M were Protein Marker (Genscript 94KD, 66kD, 36KD, 25KD, 14KD). Lanes 1-4 were detected in different E. coli transformants. expression.
  • FIG. 2 SDS-PAGE was used to detect the expression of protein A dimer in E. coli, wherein lane M was Protein Marker (Genscript 94KD, 66kD, 36KD, 25KD, 14KD), and lane 5 was to detect protein A dimer in different large intestines. Expression in Bacillus transformants.
  • FIG. 3 SDS-PAGE Protein A was purified by NI column, lane 1 was Protein Marker (Genscript 94KD, 66kD, 36KD, 25KD, 14KD), and lane 2 was the sample eluted when eluting protein A with elution buffer.
  • lane 1 was Protein Marker (Genscript 94KD, 66kD, 36KD, 25KD, 14KD)
  • lane 2 was the sample eluted when eluting protein A with elution buffer.
  • Figure 4 SDS-PAGE Purification of Protein A Dimer by NI Column
  • Lane 1 is Protein Marker (Genscript 94KD, 66kD, 36KD, 25KD, 14KD)
  • Lane 2 is the sample before the Ni column on the cell disruption supernatant.
  • Lane 3 is the efflux sample of the cell disrupted supernatant after passing through the Ni column
  • lane 4 is the sample flowing out when washing with the equilibration buffer
  • lane 5 is the sample flowing out when the protein A dimer is eluted with the elution buffer.
  • FIG. 5 SDS-PAGE Protein A as a ligand for affinity chromatography media purification of immunoglobulin
  • Lane 1 is Protein Marker (Genscript 94KD, 66kD, 36KD, 25KD, 14KD)
  • Lane 2 is human serum
  • Lane 3 is used Protein
  • FIG. 6 SDS-PAGE protein A dimer as a ligand for affinity chromatography to purify immunoglobulin
  • Lane 1 is Protein Marker (Genscript 220KD, 150KD, 100KD, 75KD, 50KD, 35kD, 25kD, 15KD), lane
  • lane 2 is human serum
  • lane 3 is a sample eluted with phosphate buffer
  • lane 4 is an immunoglobulin purified from human serum using a ligand of protein A dimer as an affinity chromatography medium.
  • Example 1 Construction of a vector encoding a protein A containing a N-terminal fusion of six histidine residues
  • the gene sequence encoding the N-terminal fusion of six histidine residues protein A was designed according to the preference of the E. coli codon and the tendency to avoid the formation of secondary structure in the mRNA coding region, as shown in SEQ ID N0.4 and SEQ ID NO. 6 shows the corresponding amino acid sequence as shown in SEQ ID N0.3 and SEQ ID N0.5, respectively, the former being referred to as protein Al and the latter as protein A2.
  • the gene coding software was used to decompose the coding sequences of the protein A1 and A2 into a plurality of small gene fragments which were bridged to each other and had similar annealing temperatures.
  • primers were synthesized according to the gene sequences of the above small fragments, such as primers 4-1 ⁇ primers4- 8 (SEQIDN0.4-1-4-8) and primer 6-1 ⁇ primer 6-8 (SEQIDN0.6-l ⁇ 6-8), with primer 4-1 ⁇ primer 4-8 and primer 6-1 ⁇
  • the mixture of primers 6-8 was a template and a primer, using PBO polymerase (genscript), using a 2720 thermal cycler (Applied Biosysytems), and the reaction cycle was 95 ° C for 20 s, 55 ° C for 20 s, and 72 ° C for 20 s.
  • Primer 6-5 AGAACAGCGTAACGCATTCATCAAGTCTATCCGCGATGATCCG Primer 6-6 CCCAGCACGTTCGTAGACTGGCTCGGATCATCGCGGATAG Primer 6-7 CTACGAACGTGCTGGGCGAAGCGAAAAAACTGAATGATGC Primer 6-8 CATATGTCATTTCGGGGCCTGCGCATCATTCAGTTTTTTCGC
  • the sequencing-verified DNA was used as a template, with primer 7 (SEQ ID N0.7) and primer 8 (SEQ ID N0.8) or primer 7 (SEQ ID N0.7) and primer 9 (SEQ ID N0.9), respectively.
  • primer 7 SEQ ID N0.7
  • primer 8 SEQ ID N0.8
  • primer 7 SEQ ID N0.7
  • primer 9 SEQ ID N0.9
  • the coding sequence of protein A 1 or protein A 2 was amplified by PCR, purified by agarose electrophoresis, and cloned using the CloneEZ Cloning Kit (Genscript), and cloned according to the product specification, and the gene sequence was integrated into the vector PET15b.
  • the vector incorporating the protein A1, 2 gene constructed in the present example is indicated by the following abbreviation: a vector containing the protein A 1 gene represented by SEQ ID N: PET15b-ProteinAl; containing the protein A represented by SEQ ID N0.5 2 gene carrier: PET15b-proteinA2.
  • Primer 13-12 using a mixture of primers 11-1 to 11-12 or primers 13-1 to 13-12 as templates and primers, using PBO polymerase (genscript), using 2720 thermal cycler (Applied Biosysytems) Company), the reaction cycle is 95 °C 20s, 55 °C 20s, 72 °C 20s, a total of 25 cycles, and finally incubated at 72 ° C for 3 minutes, to obtain a PCR reaction solution, using the secondary reaction solution as a template, add the above Primer primer 11-1 and primer 11-12 or primer 13-1 and primer 13-12 designed based on the first and last small fragment gene sequences were used as primers, and PCR was carried out once under the same conditions, and the obtained PCR product was added with ethidium bromide.
  • the 1% agarose gel was subjected to electrophoresis, and the DNA band was examined by observing the gel under ultraviolet light.
  • the amplified fragment was excised from the gel and purified using a Quick Gel Extraction Kit (Genscript) according to the manufacturer's instructions.
  • Purified DNA A DNA sequencer (3730x1 96-capillary DNA analyzer) manufactured by Applied Biosystems Co., Ltd., and ABI PRISM Bigdye Terminator Cycle Sequencing Ready Reaction Kit were used, and according to the product specification, the order was reviewed.
  • the sequencing-verified DNA was used as a template, with primer 7 (SEQ ID N0.7) and primer 14 (SEQ ID N0.14) or primer 7 (SEQ ID N0.7) and bow
  • primer 7 SEQ ID N0.7
  • primer 14 SEQ ID N0.14
  • primer 7 SEQ ID N0.7
  • 15 SEQ ID N0.15
  • the coding sequence of the protein AA1, AA2 dimer was amplified by PCR, purified by agarose electrophoresis, cloned using the CloneEZ Cloning Kit (Genscript), and cloned according to the product specification, and the gene sequence was integrated into the vector PET15b.
  • a vector incorporating the protein AA1 or AA2 gene constructed in the present example is indicated by the following abbreviations: a vector comprising the protein AA1 dimer gene of SEQ ID NO.
  • PET15b-ProteinAAl containing the protein represented by SEQ ID N0.12 Vector of the AA2 dimer gene: PET15b-ProteinAA2.
  • SEQ ID N0.12 Vector of the AA2 dimer gene: PET15b-ProteinAA2.
  • the plasmid PET15b-P r0 t e inAl containing the protein A 1 dimer gene was transfected into E. coli BL21 competent cells.
  • Escherichia coli BL21 containing the PET15b-ProteinAl plasmid was placed in culture medium (lg/L peptone, 5 g/L yeast extract, 5 g/L NaCI and 100 mg/L Ampicillin) at 37 ° C, when E. coli reached logarithmic growth
  • culture medium lg/L peptone, 5 g/L yeast extract, 5 g/L NaCI and 100 mg/L Ampicillin
  • the plasmid PET15b-proteinAAl containing the protein A 1 dimer gene was transfected into E. coli BL21 competent cells.
  • Escherichia coli BL21 containing the PET15b-proteinAAl plasmid was placed in culture medium (lg/L peptone, 5 g/L yeast extract, 5 g/L NaCI and 100 mg/L Ampicillin) at 37 ° C, when E. coli reached logarithmic growth
  • culture medium lg/L peptone, 5 g/L yeast extract, 5 g/L NaCI and 100 mg/L Ampicillin
  • Protein A or protein A dimer nitrogen-containing amino acid is used to bond to the surface of the epoxy-activated agarose, thereby preparing a protein A or protein coupled with a N-terminal fusion of six histidine residues.
  • a dimer agarose medium 10 mg of Protein A or Protein A dimer was coupled via epoxy to the surface of 1 ml of Sepharose 4B (GE Healthcare) agarose medium, and 0.5 ml of the coupled N-terminally fused 6 histidine residues were taken. The protein A or protein A dimer was detected on agarose medium. Rinse the constant flow pump with 20 ml of double distilled water, then rinse the column and add 0.5 ml of the column to the column until the column is naturally precipitated.
  • the column was washed with a constant flow pump with 10 ml of 20 mM phosphate buffer (containing 0.15 M NaCI, 30 mM Na 2 HPO 4 , 10 mM NaH 2 P0 4 adjusted to pH 7.0), and the column was equilibrated at a flow rate of 1 ml/min.
  • Serum was sampled at a concentration of 15 ml of 5 mg/ml human serum, and the sample was loaded at a flow rate of 0.5-1 ml/min to saturate the column, followed by 20 ml of 20 mM phosphate buffer (containing 0.15 M NaCI, 30 mM). Na 2 HP0 4 , 10 mM NaH 2 P0 4 was adjusted to P H 7.0) Washed, finally eluted with 0.1 M pH 3.0 glycine eluate, and the eluted target protein was collected under UV detection, and 20 uL was taken. The eluted fraction was subjected to SDS-PAGE for 4-20% gel concentration.
  • Example 8 Alkali resistance test of a protein A dimer containing a N-terminal fusion of 6 histidine residues as a ligand for an affinity chromatography medium
  • the alkaline solution was in situ cleaned and detected by using 0.5 ml of an agarose column coupled with a protein A dimer of N-terminally fused 6 histidine residues.
  • the immunoglobulin was purified according to the procedure of Example 7. After the immunoglobulin was eluted with 0.1 M pH 3.0 eluate, the alkaline solution was washed in 15 ml of 0.5 M NaOH solution at a flow rate of 1 ml.
  • the chromatographic medium was washed at a rate of /min, and the column was equilibrated with 10 ml of 20 mM phosphate buffer (containing 0.15 M NaCI, 30 mM Na 2 HP04, 10 mM NaH 2 P04 adjusted to pH 7.0) to complete an alkaline solution in situ cleaning test.
  • the loop The ability of the peptone dimer to bind to the immunoglobulin as an affinity chromatography ligand can be determined in each cycle based on the amount of immunoglobulin in the eluate.

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Abstract

本发明属于蛋白工程领域，涉及一类突变的具有高耐碱特性的蛋白A及其应用。本发明蛋白A能牢固结合到免疫球蛋白分子除互补决定区之外其他区域，可以作为偶联于固相载体的配体用于分离免疫球蛋白。这类蛋白A能在PH13-14强碱环境中保持化学稳定性，作为层析配体从而可以耐受苛刻的原位清洗条件。本发明也涉及使用蛋白A配体分离纯化免疫球蛋白和用碱再生的过程。

Description

一类突变的具有高耐碱特性的蛋白 A及其应用 技术领域
本发明属于蛋白工程领域， 涉及一类突变的具有高耐碱特性的蛋白 A及其应用。 背景技术
生物技术是当今世界高技术发展最快的领域之一。 作为生物技术领域之一的抗体药物， 近些年， 在不断地提高患者健康水平和生活质量的同时， 也取得了瞩目的市场业绩。 另外， 新药研发不断增加投入的同时， 创新药物却在明显减少， 且目前众多小分子药物还面临着专 利悬崖的威胁。 所以， 为了寻求新的增长点， 众多制药企业尤其是生物技术制药公司， 逐渐 进入抗体药物研发领域。 目前， 抗体药物在基础生物医学研究、 疾病（如癌症、 器官移植排 斥、 自身免疫性疾病等）的诊断和治疗方面已经被广泛应用。
近年来随着治疗性药用抗体药物在医疗领域大量出现，其生产工艺也开始受到人们的重 视。 对于用于药物和诊断试剂的抗体药物的大规模经济生产， 一般而言， 是通过细胞培养在 细胞内或通过分泌进入周围的培养基中生产目标抗体。 由于培养细胞的过程需要在培养基中 加入糖， 氨基酸， 生长因子， 因此必须将抗体从培养基和其他细胞组分中分离达到足够的纯 度， 才可以用作人治疗剂。 现在最普遍采用的抗体纯化方式是亲和层析， 其优势在于简单快 速选择性强， 早期先进行亲和纯化， 可以明显减少后续纯化的步骤。 在现代工业中保持低的 生产成本也是生产工艺中很重要的要求， 如果生产中所需的纯化层析介质可以反复使用， 可 以明显降低抗体的生产成本。 但是， 由于层析介质每次纯化抗体都会留下未洗脱的蛋白， 蛋 白聚集体甚至残留对人体有害的物质， 例如病毒， 内毒素。 所以介质在重新使用时必须进行 清洁， 现在最有效的恢复层析介质的清洁方式是被称作原位清洁的碱性方法， 其标准流程包 括用 0. 5M Na0H处理纯化介质， 这种苛刻的方式可以有效地去除杂质， 但是很可能损害纯化 介质。因此本发明涉及的耐碱的并可以结合免疫球蛋白的蛋白 A分子可以作为亲和介质上很 好的配体， 用于纯化抗体药物， 其纯化介质也可以用原位清洁的碱性方法对使用后的纯化介 质进行再生。 发明内容
本发明的目的是针对现有技术的上述不足， 提供一类突变的具有高耐碱特性的蛋白 A。 本发明的另一目的是提供该蛋白 A的应用。
本发明的目的可通过如下技术方案实现：
一类基因突变的蛋白 A， 其氨基酸序列如 SEQ ID N0.1或 SEQ ID N0.2所示， 或者与 SEQ ID N0.1或 SEQ ID N0.2所示的氨基酸序列在与免疫球蛋白结合区域有 99%以上同源性的能够 结合免疫球蛋白的所有氨基酸序列， 其中， 所述的与免疫球蛋白结合区域为第 7~第 54个氨 基酸残基。
编码所述的蛋白 A的核酸。
本发明涉及的蛋白 A， 实质上是一类可以结合免疫球蛋白的分子， 这类分子可以牢固结 合免疫球蛋白上互补决定区之外其他区域， 同时可以耐受 PH13-14的强碱环境。 这类蛋白 A 的结合免疫球蛋白的区域的蛋白构象主要是有 3股 α螺旋组成，并折叠成一种三螺旋束结构。 其能通过 α螺旋 1和 2表面氨基酸残基结免疫球蛋白的 Fc区域， 或通过 α螺旋 2和 3表面 残基结合免疫球蛋白 Fab上的非互补决定区。 同时 α螺旋 1， 2， 3表面疏水性残基会在螺旋 束的中心形成一个疏水核心， 可以将 α螺旋 1， 2, 3紧密的结合在一起， 维持蛋白稳定的结 构。 与其他不耐碱蛋白不同的是当这类蛋白 Α被放置于 PH13-14的碱性环境中， 虽然维持蛋 白稳定结构的氢键消失， α螺旋 1、 2、 3会舒展伸张， 但是强的疏水力仍能使 α螺旋 1、 2、 3紧密结合并保证其相对位置没有显著变化。当这类蛋白 Α重新被放置于 PH7-8中性环境中， α螺旋 1、 2、 3间氢键恢复， 因此整体构象可以恢复至放置于碱性环境之前， 而其他不耐碱 蛋白由于缺乏维持蛋白结构稳定的疏水核心， 在被放置于 PH13-14的碱性环境中后， 蛋白结 构会被彻底破坏， 当重新被放置于 ΡΗ7-8中性环境中， 蛋白整体构象无法恢复至放置于碱性 环境之前。将这种有耐碱特质的蛋白 Α及其多聚体偶联到固相载体上可以用来分离纯化免疫 球蛋白分子或者将他们偶联上标记物可以用来检测免疫球蛋白分子， 同时如果将其作为层析 介质的配体， 可以用原位碱清洁的方式对层析介质实现再生。
本发明蛋白 A单聚体分子量在 6KD左右， 由 58个氨基酸组成， 可以通过多肽化学合成 或是细胞表达的方式获得。 如果采用细胞表达的方式， 必须首先构建携带有编码蛋白氨基酸 序列的基因的细胞株。 因此需要按照以下的流程进行： 首先通过基因合成的方式获取编码蛋 白 A氨基酸序列的基因， 下一步， 将编码蛋白 A氨基酸序列的基因重组到表达载体中， 此时 可以根据需要在载体中添加纯化标签筛选标签或是指示性标签， 紧接着将表达载体稳定地转 入合适的细胞中， 完成整个可以生产这类蛋白 A的表达***。 所需要的具有结合免疫球蛋白 分子的蛋白 A (包括单聚体或多聚体）可以从培养基或者细胞中得到。 必须注意的是蛋白 A 在细胞中的表达需要受到紧密的控制， 因此选择用于表达蛋白 A的载体非常重要， 应该具有 以下几个特性： 1.表达载体含有启动子或初始转录位点， 2 表达载体含有操纵子用来开关基 因的表达 ， 3.表达载体含有核糖体结合位点， 4. 表达载体含有转录和翻译的终止位点， 可 以提高转录和翻译产物的稳定性。 因此可以推荐表达蛋白 A的载体有 PET (Novagen) , PQE30(Qiagen), PGS21a (genscript) , pGAPZa A ( Invitrogen )等， 对应的宿主细胞的选择有大 肠杆菌基因工程菌和毕赤巴斯德酵母， 可以在宿主的细胞内， 细胞外或细胞膜上生成这类蛋 S A。
这类蛋白 A可以根据需要， 通过利用其物理性质， 化学性质等的各种分离操作进行分离 纯化。 具体而言， 例如通常的蛋白沉淀剂处理 （盐析法）， 离心分离， 渗透压破碎法， 超声 波破碎， 超滤， 凝胶过滤， 以及吸附色谱， 离子交换色谱， 亲和色谱， 高速液相色谱等各种 液相色谱，透析法及这些方法的组合等。此外，通过表达这类蛋白与亲和标记融合的蛋白质， 利用该标记可以进行亲和纯化。 此处所述的亲和标记， 例如多聚组氨酸标记及 FLAG标记。 利用这些标记可以进行这类蛋白 A的纯化。
这类蛋白 A结合免疫球蛋白的活性可以通过 ELISA实验测定。 例如将这类蛋白 A固定在 固相载体上， 然后加入酶标记的免疫球蛋白使其吸附在蛋白 A上， 用洗涤的方法使固相载体 上形成的免疫球蛋白和蛋白 A的复合体与液体中其他物质分开， 这样固相载体上的蛋白 A与 免疫球蛋白形成的复合体的量与酶的量呈一比一的比例。 加入酶的反应底物后， 底物被酶催 化成为有色产物， 这样蛋白 A结合的免疫球蛋白的量可以根据呈色的深浅进行定性和定量分 析。
这类蛋白 A的碱化学稳定性也可以被本领域的技术人员很容易的证实。例如将这类蛋白 A先浸泡在 0.5M浓度的 NaOH中持续 60小时， 按照以上所述的 ELISA实验测定其结合免疫 球蛋白的活性， 如何仍然可以很好的保持结合免疫球蛋白的活性， 那么则证明这类蛋白 A在 强碱环境中是稳定的。再例如利用存在于这类蛋白 A中的氨基或羧基基团，通过双环氧化物， 表氯醇，溴化氰， N-羟基琥珀酰胺等偶联剂，将这类蛋白 A偶联到固相载体上（Sepha r0Se 4B) 并制成层析柱， 然后过量加载免疫球蛋白分子， 接着用 PH8.0的磷酸盐缓冲液洗涤， 然后用 PH.3.0的甘氨酸缓冲液洗脱， 之后测定洗脱的免疫球蛋白的量来测定柱的总容量。 在每一个 循环之间， 用 0.5M NaOH组成的原位清洁的碱性方法处理来处理柱， 在 100个循环之后， 如 果柱的总容量没有降低， 那么则证明这类蛋白 A在强碱环境下的化学性质非常稳定， 非常适 合作为免疫球蛋白亲和纯化的配体。 一种包含所述的蛋白 A的多聚体， 包含两个或两个以上重复单元。
上述论述的蛋白 A都是单聚体， 然而这种蛋白可以结合为多聚体蛋白， 诸如二聚体， 三 聚体， 四聚体等等。 因此一个属于本发明的蛋白 A与自身或与本发明中其他蛋白 A形成的多 聚体， 同样属于本发明的另一个方面。 本发明的多聚体包括优选 4-10 个氨基酸的一段氨基 酸序列连接的单体单元。 这种连接不破坏蛋白 A的空间构象， 此外在碱性环境中同样充分稳 定， 不损害蛋白 A的特性。在目前的实施方案中， 多聚体是蛋白 A的二聚体,其中连接蛋白 A 的长度为 4个氨基酸 （ADGK)。
本发明涉及编码上述蛋白 A或其多聚体的核酸序列。 该核酸序列经过密码子优化， 优化 后的核酸序列避免了稀有密码子的产生和二级结构的形成， 并通过引物搭桥人工基因合成的 方式合成出来。
本发明涉及这类蛋白 A经 N端或 C端或侧链基团进行化学修饰所得到的所有能牢固结合 免疫球蛋白的蛋白衍生物。 如 N端氨基被乙酰化后得到 N-乙酰多肽， 被脂肪酸酰化后得到 N-脂肪酸酰化多肽。 又比如 C-端羧基转化为酰胺可以得到多肽酰胺， 转化为酯得到多肽酯， 转化为硫酯得到多肽硫酯。 衍生化修饰对调节原有蛋白的酸碱性， 稳定性， 溶解性， 反应活 性及生物活性具有重要作用。 如果在蛋白 A的 C末端提供一个半胱氨酸残基， 蛋白 A可以通 过硫醚键与载体相偶联， 而且这种偶联方法技术人员利用标准技术和设备很容易进行。
本发明所述的蛋白 A、 多聚体或蛋白 A衍生物在分离、 纯化和 /或检测免疫球蛋白中的 应用。
编码本发明所述的蛋白 A、 多聚体或蛋白 A衍生物的基因在分离、 纯化和 /或检测免疫 球蛋白中的应用。
本发明涉及这类蛋白 A的商业化应用， 包括免疫球蛋白的纯化和免疫球蛋白的检测。 免疫球蛋白纯化方面的应用包含一种层亲和析分离的方法，其中通过吸附于如上所述蛋白 A 或多聚体来从复杂基质中分离至少一种靶化合物， 是一种广泛使用的分离技术。 具体步骤包 括让含有免疫球蛋白的样品溶液流经蛋白 A层析介质。在此步骤中， 溶液中的其他组分将基 本不受阻碍地通过， 而免疫球蛋白被吸附到层析介质上。 然后洗涤介质， 例如用磷酸盐缓冲 液， 来除去残留的非特异性结合的杂质， 正如以上所讨论的， 也可以通过利用碱性试剂的洗 涤步骤。 下一步骤中， 让洗脱液流经层析介质从而将吸附的免疫球蛋白洗脱下来， 洗脱通 常是通过改变 PH， 离子强度或是添加竞争性的物质来实现的。 免疫球蛋白的检测方面的应 用包含一种检测免疫球蛋白的方法， 其中包括首先在蛋白 A上进行酶标记， 化学发光标记或 是同位素标记， 然后通过蛋白 A吸附到免疫球蛋白上， 能够实现免疫球蛋白可视化分析。 有益效果：
本发明提供的蛋白 A 及其多聚体和衍生物能牢固结合到免疫球蛋白分子除互补决定区 之外其他区域， 可以作为偶联于固相载体的配体用于分离免疫球蛋白。 这类蛋白 A 能在 PH13-14强碱环境中保持化学稳定性， 作为层析配体从而可以耐受苛刻的原位清洗条件。 本 发明蛋白 A及其多聚体和衍生物能够在纯化和 /或检测免疫球蛋白中应用。 附图说明
图 1 SDS-PAGE检测蛋白 A在大肠杆菌中的表达， 其中泳道 M为 Protein Marker (Genscript 94KD， 66kD， 36KD, 25KD, 14KD )， 泳道 1~4分别为检测蛋白 A在不同大肠杆菌转换子 中的表达。
图 2 SDS-PAGE 检测蛋白 A二聚体在大肠杆菌中的表达， 其中泳道 M 为 Protein Marker (Genscript 94KD, 66kD， 36KD, 25KD, 14KD )， 泳道 5分别为检测蛋白 A二聚体在不 同大肠杆菌转换子中的表达。
图 3 SDS-PAGE 用 NI柱纯化蛋白 A， 其中泳道 1为 Protein Marker (Genscript 94KD, 66kD， 36KD, 25KD, 14KD )， 泳道 2为用洗脱缓冲液洗脱蛋白 A时流出的样品。
图 4 SDS-PAGE 用 NI柱纯化蛋白 A二聚体， 其中泳道 1为 Protein Marker (Genscript 94KD, 66kD， 36KD, 25KD, 14KD )， 泳道 2为细胞破碎液上清上 Ni柱前的上样样品， 泳道 3为 细胞破碎液上清经过 Ni柱后的流出样品， 泳道 4为用平衡缓冲液洗杂时流出的样品， 泳道 5 为用洗脱缓冲液洗脱蛋白 A二聚体时流出的样品。
图 5 SDS-PAGE蛋白 A作为亲和层析介质的配体纯化免疫球蛋白，其中泳道 1为 Protein Marker (Genscript 94KD, 66kD， 36KD, 25KD, 14KD )， 泳道 2为人的血清， 泳道 3为用以蛋白
A作为亲和层析介质的配体从人的血清中纯化出的免疫球蛋白。
图 6 SDS-PAGE蛋白 A二聚体作为亲和层析介质的配体纯化免疫球蛋白，其中泳道 1为 Protein Marker (Genscript 220KD, 150KD, 100KD, 75KD, 50KD, 35kD, 25kD, 15KD ), 泳道
2为人的血清， 泳道 3为用磷酸盐缓冲液洗杂时流出的样品， 泳道 4为以蛋白 A二聚体作为 亲和层析介质的配体从人的血清中纯化出的免疫球蛋白。
图 7蛋白 A二聚体作为亲和层析介质的配体的耐碱性测试。 具体实施方式
实施例 1 含有 N末端融合 6个组氨酸残基的蛋白 A的编码基因的载体的构建
根据大肠杆菌密码子的偏好性以及避免 mRNA编码区形成二级结构的倾向设计出编码 N 端融合 6个组氨酸残基蛋白 A的基因序列， 分别如 SEQ ID N0.4和 SEQ ID N0.6所示； 其对应 的氨基酸序列分别如 SEQ ID N0.3和 SEQ ID N0.5所示，前者以下称为蛋白 Al，后者称为蛋白 A2。运用基因设计软件将蛋白 A1和 A2的编码基因序列分解成多条相互搭桥且退火温度相差 不大的基因小片段， 根据上述小片段的基因序列合成引物， 分别如引物 4-1~引物 4-8 ( SEQIDN0.4-1-4-8 ) 和引物 6-1~引物 6-8 ( SEQIDN0.6-l~6-8), 以引物 4-1~引物 4-8和引物 6-1~引物 6-8 的混合物为模版和引物， 使用 PBO 聚合酶 (genscript) , 采用 2720 thermal cycler(Applied Biosysytems公司）， 反应循环为 95°C 20s,55 °C 20s,72°C 20s,共进行 25个循环， 最后于 72°C保温 3分钟， 得到 PCR反应液； 以此次反应液为模板， 添加上述根据首尾小片 段基因序列设计的引物 4-1和引物 4-8或引物 6-1和引物 6-8作为引物，于同样的条件进行 1 次 PCR,得到的 PCR产物用加入溴乙锭的 1%琼脂糖凝胶进行电泳， 通过在紫外线下观察该凝 胶研究 DNA条带。 从凝胶中切出扩增的片段， 使用 Quick Gel Extraction Kit (Genscript)按照产 品说明书进行纯化。 纯化后的 DNA使用 Applied Biosystems 公司制的 DNA测序仪 (3730x1 96-capillary DNA analyzer)以及 ABI PRISM Bigdye Terminator Cycle Sequencing Ready Reaction Kit 并按照产品说明书进行测序。
表 1 PCR扩增蛋白 A1和蛋白 A2的引物
Figure imgf000008_0001
引物 6-2 ATGCGTTCTGTTGTTC 1 1 1 1 1 CAAACTTGCCATCTGCGCC
引物 6-3 AAAGAACAACAGAACGCATTCTACGAAATCCTGCATCTGCCGA
引物 6-4 TGCGTTACGCTGTTCTTCGGTCAGGTTCGGCAGATGCAGGA
引物 6-5 AGAACAGCGTAACGCATTCATCAAGTCTATCCGCGATGATCCG 引物 6-6 CCCAGCACGTTCGTAGACTGGCTCGGATCATCGCGGATAG 引物 6-7 CTACGAACGTGCTGGGCGAAGCGAAAAAACTGAATGATGC 引物 6-8 CATATGTCATTTCGGGGCCTGCGCATCATTCAGTTTTTTCGC
以测序验证后的 DNA为模板， 分别以引物 7 ( SEQ ID N0.7) 和引物 8 ( SEQ ID N0.8) 或 引物 7 ( SEQ ID N0.7 )和引物 9 ( SEQ ID N0.9 ) 为引物， 通过 PCR扩增蛋白 A 1或蛋白 A 2的 编码序列， 琼脂糖电泳后进行纯化， 使用 CloneEZ克隆试剂盒 (Genscript公司),并按照产品说 明书进行克隆,将基因序列整合到载体 PET15b。 整合本实施例构建的蛋白 A l， 2基因的载体 用以下略称表示：含有 SEQ ID N0.3所示的蛋白 A 1基因的载体： PET15b-ProteinAl;含有 SEQ ID N0.5所示的蛋白 A 2基因的载体： PET15b- ProteinA2。
实施例 2 含有 N末端融合 6个组氨酸残基的蛋白 A二聚体的编码基因的载体的构建
根据大肠杆菌密码子的偏好性以及避免 mRNA编码区形成二级结构的倾向设计出编码 N 端融合 6个组氨酸残基蛋白 A二聚体的基因序列，分别如 SEQ ID NO.ll或 SEQ ID N0.13所示； 其对应的氨基酸序列分别入 SEQ ID NO.10和 SEQ ID N0.12所示， 前者以下称为蛋白 AA1， 后 者称为蛋白 AA2。 运用基因设计软件将基因序列分解成多条相互搭桥且退火温度相差不大的 基因小片段， 根据上述小片段的基因序列合成引物， 分别如引物 11-1~引物 11-12和或引物 13-1~引物 13-12， 分别以引物 11-1~引物 11-12或引物 13-1~引物 13-12的混合物为模版和引 物， 使用 PBO聚合酶 (genscript) , 采用 2720 thermal cycler(Applied Biosysytems公司）， 反应循 环为 95 °C 20s,55°C 20s,72°C 20s,共进行 25个循环， 最后于 72°C保温 3分钟， 得到 PCR反应 液，以次反应液为模板，添加上述根据首尾小片段基因序列设计的引物引物 11-1和引物 11-12 或引物 13-1和引物 13-12作为引物， 于同样的条件进行 1次 PCR,得到的 PCR产物用加入溴 乙锭的 1%琼脂糖凝胶进行电泳， 通过在紫外线下观察该凝胶研究 DNA条带。 从凝胶中切出 扩增的片段，使用 Quick Gel Extraction Kit (Genscript)按照产品说明书进行纯化。纯化后的 DNA 使用 Applied Biosystems公司制的 DNA测序仪 (3730x1 96-capillary DNA analyzer)以及 ABI PRISM Bigdye Terminator Cycle Sequencing Ready Reaction Kit,并按,照产品说明书进亍观 Ij序。
表 2 PCR扩增蛋白 A l和蛋白 A2二聚体的引物
Figure imgf000010_0001
以测序验证后的 DNA为模板， 以引物 7 ( SEQ ID N0.7) 和引物 14 ( SEQ ID N0.14) 或引 物 7 ( SEQ ID N0.7) 和弓 |物 15 ( SEQ ID N0.15 ) 为引物， 通过 PCR扩增蛋白 AA1， AA2二聚 体的编码序列， 琼脂糖电泳后进行纯化， 使用 CloneEZ克隆试剂盒 (Genscript公司),并按照产 品说明书进行克隆,将基因序列整合到载体 PET15b。 整合本实施例构建的蛋白 AA1或 AA2基 因的载体用以下略称表示： 含有 SEQ ID NO.10 所示的蛋白 AA1 二聚体基因的载体： PET15b-ProteinAAl； 含有 SEQ ID N0.12 所示的蛋白 AA2 二聚体基因的载体： PET15b-ProteinAA2。 实施例 3含有 N末端融合 6个组氨酸残基的蛋白 A的表达
将含有蛋白 A 1二聚体基因的质粒 PET15b-Pr0teinAl转染大肠杆菌 BL21感受态细胞。将 含有 PET15b-ProteinAl质粒的大肠杆菌 BL21放入培养基 (lg/L蛋白胨， 5g/L酵母膏， 5g/L NaCI 和 100mg/L Ampicillin)在 37°C进行培养， 当大肠杆菌到达对数生长曲线时， 添加 0.5mM IPTG 进行诱导蛋白表达 4个小时， 通过离心收集诱导后的大肠杆菌。 取少量菌体用高温 (95 °C )进 行裂解释放出全菌蛋白， 将全菌蛋白进行 4~20%胶浓度的 SDS-PAGE。如图 1显示， 蛋白 A在 7~8KD附近检出一个条带。 实施例 4含有 N末端融合 6个组氨酸残基的蛋白 A二聚体的表达
将含有蛋白 A 1二聚体基因的质粒 PET15b- ProteinAAl转染大肠杆菌 BL21感受态细胞。 将含有 PET15b- ProteinAAl质粒的大肠杆菌 BL21放入培养基 (lg/L蛋白胨， 5g/L酵母膏， 5g/L NaCI和 100mg/L Ampicillin)在 37°C进行培养， 当大肠杆菌到达对数生长曲线时， 添加 0.5mM IPTG进行诱导蛋白表达 4个小时，通过离心收集诱导后的大肠杆菌。取少量菌体用高温 (95 °C ) 进行裂解释放出全菌蛋白， 将全菌蛋白进行 4~20%胶浓度的 SDS-PAGE。如图 2显示， 蛋白 A 二聚体在 14~15KD附近检出一个条带。 实施例 5含有 N末端融合 6个组氨酸残基的蛋白 A的纯化
用蒸馏水把恒流泵冲洗干净,然后把玻璃层析柱冲洗干净。 向柱中加入约 200ml Ni-IDA ( Genscript) 装柱， 待柱料全部自然沉淀下来。 通过恒流泵用平衡缓冲液 (20mM Tris 300mM NaCI)平衡约 3L， 流速为 5ml/min ; 将 lOg诱导后的含有蛋白 A 1的大肠杆菌用 200ml平衡缓 冲液 (20mM Tris 300NaCI)重悬， 将细胞用超声破碎仪 （宁波新芝生物有限公司」 Y98-IIIDH ) 破 碎， 将破碎液离心后的上清上样， 流速为 2ml/min; 上样完后， 用平衡缓冲液洗杂至吸光度 不变，流速 5ml/min ;洗杂完毕后，用 Elution buffer(20mM Tris ,300mM NaCI,250mM Iminazole) 开始洗脱， 流速 5ml/min， 收集洗脱液， 将其进行 4~20%胶浓度的 SDS-PAGE检测。 如图 3 显示， 蛋白 A经纯化后纯度达到 90%以上。 实施例 6含有 N末端融合 6个组氨酸残基的蛋白 A二聚体的纯化
用蒸馏水把恒流泵冲洗干净,然后把玻璃层析柱冲洗干净。 向柱中加入约 200ml Ni-IDA ( Gensc pt) 装柱， 待柱料全部自然沉淀下来。 通过恒流泵用平衡缓冲液 (20mM Tris 300mM NaCI)平衡约 3L，流速为 5ml/min ;将 lOg诱导后的含有蛋白 AA1的大肠杆菌用 200ml平衡缓 冲液 (20mM Tris 300NaCI)重悬， 将细胞用超声破碎仪 （宁波新芝生物有限公司」 Y98-IIIDH ) 破 碎， 将破碎液离心后的上清上样， 流速为 2ml/min; 上样完后， 用平衡缓冲液洗杂至吸光度 不变，流速 5ml/min ;洗杂完毕后，用 Elution buffer(20mM Tris ,300mM NaCI,250mM Iminazole) 开始洗脱， 流速 5ml/min， 收集洗脱液， 将其进行 4~20%胶浓度的 SDS-PAGE检测。 如图 4 显示， 蛋白 A二聚体经纯化后纯度达到 90%以上。 实施例 7 将含有 N末端融合 6个组氨酸残基的蛋白 A或蛋白 A二聚体作为亲和层析介质的 配体纯化免疫球蛋白
利用蛋白 A或蛋白 A二聚体含氮基的氨基酸， 将其键合到环氧活化的琼脂糖表面， 从而制备得偶 联有含有 N末端融合 6个组氨酸残基的蛋白 A或蛋白 A二聚体的琼脂糖介质。 将 10mg蛋白 A 或蛋白 A二聚体通过环氧基偶联到 1ml Sepharose 4B (GE Healthcare)琼脂糖介质的表面，并取其中 0.5ml的偶联有含有 N末端融合 6个组氨酸残基的蛋白 A或蛋白 A二聚体的琼脂糖介质进行 检测。 先用 20ml 双蒸水把恒流泵冲洗干净， 然后把柱子冲洗干净， 向柱子里加入 0.5ml的 柱料， 待柱料全部自然沉淀下来。 通过恒流泵用 10ml 20mM磷酸盐缓冲液 (含有 0.15M 的 NaCI ,30mM Na2HPO4 ,10mM NaH2P04调为 pH 7.0)冲洗柱料,平衡柱料， 流速为 lml/min。
以体积 15ml浓度为 5mg/ml人的血清为检测样品， 以流速 0.5-1 ml/min的速度上样， 让 柱料吸附饱和， 再用 20ml 20mM磷酸盐缓冲液 (含有 0.15M的 NaCI ,30mM Na2HP04 ,10mM NaH2P04调为 PH 7.0)洗杂， 最后用 0.1M pH 3.0的甘氨酸洗脱液洗脱目的蛋白，在紫外检测下 收集洗脱下来的目的蛋白，取 20uL的洗脱组分进行 4~20%胶浓度的 SDS-PAGE检测。如图 5,6 显示， 蛋白 A或蛋白 A二聚体可以分离出高纯度的免疫球蛋白。 实施例 8含有 N末端融合 6个组氨酸残基的蛋白 A二聚体作为亲和层析介质的配体的耐碱性 测试
用 0.5ml的偶联有含有 N末端融合 6个组氨酸残基的蛋白 A二聚体的琼脂糖柱子进行碱 溶液原位清洗检测。先按照实施例 7的步骤进行免疫球蛋白的纯化，在免疫球蛋白被 0.1M pH 3.0的甘氨酸洗脱液洗脱后， 以 15ml 0.5M NaOH溶液为原位清洗的碱溶液， 以流速 1 ml/min 的速度清洗层析介质， 再用 10ml 20mM 磷酸盐缓冲液（含有 0.15M 的 NaCI ,30mM Na2HP04 ,10mM NaH2P04调为 pH 7.0)平衡柱料， 完成一个碱溶液原位清洗检测的循环。 在 每个循环中可以根据洗脱液中的免疫球蛋白的量确定作为亲和层析配体的蛋白 Α 二聚体结 合免疫球蛋白的能力。
如图 7显示，作为亲和层析配体的蛋白 A二聚体经过 100个碱溶液原位清洗检测的循环 仍然保持良好的结合免疫球蛋白的能力。

Claims

权利要求书
1. 一类基因突变的蛋白 A，其特征在于其具有如 SEQ ID N0.1或 SEQ ID N0.2所示氨基酸序列， 或者与 SEQ ID N0.1或 SEQ I D N0.2所示的氨基酸序列在与免疫球蛋白结合区域有 99%以上 同源性的能够结合免疫球蛋白的所有氨基酸序列， 其中， 所述的与免疫球蛋白结合区域为 第 7~第 54个氨基酸残基。
2. 一种编码权利要求 1所述的蛋白 A的基因。
3. 一种包含权利要求 1所述的蛋白 A的多聚体，其特征在于所述的多聚体为两个或两个以上 权利要求 1所述的蛋白 A形成的多聚体或者为权利要求 1所述的蛋白 A与其他蛋白形成的 多聚体。
4. 一种编码权利要求 3所述的多聚体的基因。
5. 对权利要求 1或 3中任一项所述的蛋白 A或蛋白 A的多聚体的 N端或 C端或侧链基团进行 化学修饰所得到的所有能牢固结合免疫球蛋白的蛋白 A衍生物。
6. 根据权利要求 5所述的蛋白 A衍生物， 其特征在于所述的衍生物是在权利要求 1或 3中任 一项所述的蛋白 A分子的 N端修饰 6个组氨酸残基所得。
7. 权利要求 1所述的蛋白 A、权利要求 3所述的多聚体或权利要求 5所述的蛋白 A衍生物在 分离、 纯化和 /或检测免疫球蛋白中的应用。
8. 根据权利要求 7所述的应用， 其特征在于包括将所述的蛋白 A、 所述的多聚体或所述的蛋 白 A衍生物用于亲和层析介质中的配体或用于免疫球蛋白检测中的分子标记物。
9. 权利要求 2、权利要求 4或权利要求 8所述的基因在分离、纯化和 /或检测免疫球蛋白中的 应用。
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