CN105481954B - Recombinant protein A and application thereof - Google Patents
Recombinant protein A and application thereof Download PDFInfo
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- CN105481954B CN105481954B CN201410471277.2A CN201410471277A CN105481954B CN 105481954 B CN105481954 B CN 105481954B CN 201410471277 A CN201410471277 A CN 201410471277A CN 105481954 B CN105481954 B CN 105481954B
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Abstract
The invention relates to a recombinant protein A and application thereof. Specifically, the invention discloses a novel recombinant staphylococcus aureus protein A, the structural description of which is detailed in the specification, the function of which comprises but is not limited to being as a ligand to specifically bind immunoglobulin, the recombinant protein A can be used as a specific affinity ligand and has excellent alkali resistance. The invention also relates to a method for constructing the mutant protein.
Description
Technical Field
The invention belongs to the field of biotechnology. Specifically, the invention relates to a novel recombinant staphylococcus aureus protein A and application thereof in protein separation and purification.
Background
Staphylococcus aureus Protein A (SpA) includes 5 domains, E, D, A, B, C, wherein the B domain (fragment B) binds most strongly to binding immunoglobulins. Wild-type staphylococcal protein a as well as recombinant staphylococcal protein a (rpa) and modified proteins have been widely used in the field of affinity chromatography, and affinity chromatography media made from staphylococcal protein a are widely used to capture antibodies, remove host proteins and residual DNA.
Affinity chromatography media often require washing in order to preserve the selectivity and high specificity of the affinity ligand and to prevent bacterial contamination, typically with 0.1-0.5M NaOH solution, with a pH in the range of 12-14. Because the protein is easy to denature under the alkaline condition, the active function is lost. Therefore, the affinity chromatography medium is subjected to alkali liquor in-situ cleaning, and after multiple cycles, the binding force of the medium to the target molecule is obviously lost, so that the purification cost is increased, and the purity of the target product is reduced due to the falling off of the protein A.
The B fragment of S.aureus protein A was engineered as a Z fragment with The glycine at position 29 replaced by alanine (Stahl et al, 1999.Affinity fusion in biotechnology: focus on protein A and protein G, in The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioclassification. M.C.Fleckinger and S.W.Drew, eds. John Wiley and Sons Inc., New York, 8-22). The Z fragment, which is used as a ligand In affinity chromatography, shows enhanced stability to certain chemicals other than NaOH compared to the B fragment, but is still unstable under higher alkaline conditions and cannot meet the need for industrial on-line Cleaning (CIP) regeneration. Therefore, there is still a need in the art to develop a protein a that is more stable, in particular more alkali resistant.
Disclosure of Invention
It is an object of the present invention to provide a novel recombinant staphylococcus aureus protein a-mer that functions including, but not limited to, specifically binding immunoglobulins as a ligand.
In a first aspect of the invention there is provided a recombinant protein a-mer which is a multimer of two or more Z-segments of staphylococcus aureus protein a in tandem, and at least one of the Z-segments is a mutant Z-segment having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, each of said Z segments is a Z segment mutant having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, the recombinant protein A mer is a 2-20 mer, preferably a 4-10 mer.
In another preferred embodiment, the recombinant protein A polymer is a 4-polymer, a 6-polymer, an 8-polymer or a 10-polymer.
In another preferred embodiment, two adjacent Z segments in the recombinant protein A mer are directly connected or connected through a connecting peptide.
In another preferred embodiment, the recombinant protein a mer consists of a Z segment and a linker peptide; the linker peptide is present between the Z segments, and the Z segments are all Z segment mutants with the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, the recombinant protein A-mer has the structure of formula (I):
Pa-(Za-L)n-Za-Pb (I)
in the formula (I), the compound is shown in the specification,
pa is null, Met, leader sequence, secretory peptide sequence, tag sequence for purification, or a combination thereof;
each Za is independently an amino acid sequence of a Z fragment of staphylococcus aureus protein a, and at least one Za is a Z fragment mutant having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr;
l is independently nothing, or a linker peptide sequence;
n is a positive integer not less than 1;
pb is nothing, Cys, a purified tag sequence, or a combination thereof.
In another preferred embodiment, n is a positive integer from 1 to 50, preferably from 2 to 20, preferably 2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, all Za are Z fragment mutants having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, the sequence of Za is shown in SEQ ID No. 3.
In another preferred embodiment, L is a linker peptide sequence of 3-50 (preferably 3-20) amino acids in length.
In another preferred embodiment, L is selected from the group consisting of: QKDAVFP, asttkgp.
In another preferred embodiment, the recombinant protein a-mer has the structure of formula (II):
Met-Za-L-Za-L-Za-L-Za-Cys (II)
wherein Za and L are as defined above.
In another preferred embodiment, the recombinant protein a-mer is a protein having antibody binding ability.
In another preferred embodiment, the antibody comprises an IgG antibody, an IgM antibody, an IgA antibody.
In another preferred embodiment, the C-terminus of the recombinant protein a-mer further comprises the amino acid residue Cys.
In another preferred example, the recombinant protein a-mer has an amino acid sequence as set forth in SEQ ID No.: 5.
The present invention provides in a second aspect a polynucleotide encoding a recombinant protein a-mer according to the first aspect of the invention.
In the case of a known protein amino acid sequence, one skilled in the art can design an appropriate polynucleotide sequence encoding the amino acid sequence as desired and express it.
In another preferred embodiment, the polynucleotide has a sequence as set forth in SEQ ID No.: 1 is shown.
In a third aspect, the present invention provides a vector comprising a polynucleotide according to the second aspect of the invention.
In a fourth aspect, the present invention provides a host cell comprising a vector according to the third aspect of the present invention or a genome thereof into which a polynucleotide according to the second aspect of the present invention has been integrated.
In a fifth aspect, the present invention provides a method of producing a recombinant protein a-mer of the first aspect of the invention, comprising the steps of:
(a) culturing the host cell of the fourth aspect of the invention, thereby expressing the recombinant protein a-mer of the first aspect of the invention; and
(b) isolating or purifying the recombinant protein A-mer from the culture system.
In a sixth aspect, the present invention provides an affinity chromatography medium comprising a recombinant protein a mer according to the first aspect of the invention.
In another preferred embodiment, in the affinity chromatography medium, the recombinant protein a-mer is immobilized or bound to a support.
In another preferred embodiment, the recombinant protein a-mer is conjugated to the carrier via thioether-bond coupling.
A seventh aspect of the invention provides a purification method comprising the steps of: affinity chromatography is carried out using the affinity chromatography medium according to the sixth aspect of the present invention for the starting material to be purified.
In another preferred embodiment, the purification is for purifying the antibody.
In another preferred embodiment, the raw material to be purified is a liquid raw material (e.g., a culture solution, a fermentation solution, etc.) containing the antibody to be purified.
The recombinant protein A has higher alkali resistance, and the stability and the maximum carrying capacity of the protein in the affinity chromatography process can be improved by adopting the recombinant protein A.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a map of expression plasmid pET30 a-4Z'.
FIG. 2 is an SDS-PAGE electrophoresis of IPTG-induced expression of recombinant protein A mer in E.coli BL21(DE3) containing plasmid pET30 a-4Z', wherein the lanes are: 1: before induction, 2: 2hr after induction, 3: 3hr after induction.
FIG. 3 is an elution chromatogram of an SP chromatography medium capturing a recombinant protein A-mer.
FIG. 4 shows the results of the alkali resistance test of the recombinant protein A-mer and the recombinant wild-type protein A (rPA) of the present invention. Wherein, the diagram (a) is an SDS-PAGE electrophoresis diagram of the recombinant protein A polymer of the invention after being incubated at 37 ℃ for different periods of time under alkaline conditions, wherein the incubation time of each lane sample is respectively as follows: 1: 0hr, 2: 4hr, 3: 8hr, 4: 16hr, 5: 24 hr; FIG. (b) is a SDS-PAGE of recombinant wild-type protein A after incubation at 37 ℃ for various periods of time under alkaline conditions, wherein the incubation time for each lane sample is: 1: 0hr, 2: 4hr, 3: 8hr, 4: for 16 hr.
Detailed Description
The present inventors have made extensive and intensive studies and have unexpectedly found that the alkali resistance can be significantly improved by modifying the amino acid sequence of wild-type staphylococcus aureus protein a (spa), i.e., by mutating the amino acid at a specific position to a specific amino acid. In addition, compared with wild SpA, the SpA recombinant protein (recombinant protein A polymer) has better stability under alkaline conditions and higher protein loading in the affinity chromatography process, thereby optimizing the protein purification process. The present invention has been completed based on this finding.
The term "protein having an antibody-binding ability" may refer to a protein capable of binding to an Fc fragment of an immunoglobulin. However, it is not excluded that proteins binding to the Fc fragment may also bind to other regions, e.g. the Fab region of an immunoglobulin.
In the present invention, mutations are defined by the numbering of the crossover positions, preceded by wild-type or non-mutated amino acids, followed by mutated amino acids. For example, a mutation of asparagine to aspartic acid at position 3 is denoted as "Asn 3 Asp".
The term "Z fragment" refers to a protein obtained by changing amino acid 29 (glycine) of wild-type SpA (E, D, A, B, C) B fragment into alanine, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.
Recombinant protein A-mers
The invention provides a recombinant protein A-mer which is a multimer formed by the concatenation of two or more Z fragments of Staphylococcus aureus protein A, and at least one of each Z fragment is a mutant Z fragment having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, each of said Z segments is a Z segment mutant having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, the recombinant protein A mer is a 2-20 mer, preferably a 4-10 mer.
In another preferred embodiment, the recombinant protein A polymer is a 4-polymer, a 6-polymer, an 8-polymer or a 10-polymer.
In another preferred embodiment, two adjacent Z segments in the recombinant protein A-mer are directly linked or linked through a linker peptide.
In another preferred embodiment, the recombinant protein a mer consists of a Z segment and a linker peptide; the linker peptide is present between the Z segments, and the Z segments are all Z segment mutants with the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, the recombinant protein A-mer has the structure of formula (I):
Pa-(Za-L)n-Za-Pb (I)
in the formula (I), the compound is shown in the specification,
pa is null, Met, leader sequence, secretory peptide sequence, tag sequence for purification, or a combination thereof;
za is independently the amino acid sequence of a Z fragment of staphylococcus aureus protein a, and at least one Za is a Z fragment mutant with the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr;
l is independently nothing, or a linker peptide sequence;
n is a positive integer not less than 1;
pb is nothing, Cys, a purified tag sequence, or a combination thereof.
In another preferred embodiment, n is a positive integer from 1 to 50, preferably from 2 to 20.
In another preferred embodiment, all Za are Z fragment mutants having the following amino acid mutations: asn3Asp, Asn6Leu and Asn23 Thr.
In another preferred embodiment, the sequence of Za comprises the sequence shown in SEQ ID No. 3.
In another preferred example, the sequence of Za has the sequence shown in SEQ ID No. 3.
In another preferred embodiment, L is a linker peptide sequence of 3-50 (preferably 3-20) amino acids in length.
In another preferred embodiment, said L is composed of amino acids that do not or do not significantly affect the correct folding and conformation of the protein, such as glycine, alanine, serine, etc.
In another preferred embodiment, L is selected from the group consisting of: QKDAVFP, asttkgp.
In another preferred embodiment, the recombinant protein a-mer has the structure of formula (II):
Met-Za-L-Za-L-Za-L-Za-Cys (II)
wherein Za and L are as defined above.
In another preferred embodiment, the recombinant protein a-mer is a protein having antibody binding ability.
In another preferred embodiment, the antibody comprises an IgG antibody (e.g., IgG1, IgG2, IgG3, IgG4), an IgM antibody, an IgA antibody.
In another preferred embodiment, the C-terminus of the recombinant protein a-mer further comprises the amino acid residue Cys.
In another preferred example, the recombinant protein a-mer has an amino acid sequence as set forth in SEQ ID No.: 5.
The recombinant protein A polymer of the invention not only refers to the protein which has the activity of the recombinant protein A with the antibody binding capacity and has an amino acid sequence shown as SEQ ID NO.5, but also comprises a variant form of the SEQ ID NO.5 sequence which has the same or similar function with the recombinant protein A. These variants include (but are not limited to): several (usually 1 to 20, more preferably 1 to 10, or 1 to 8, or 1 to 5, or 1 to 3, or 1 to 2) amino acids are deleted, inserted and/or substituted, and one or several (usually 1 to 20, more preferably 1 to 10, or 1 to 5) amino acids are added or deleted at the C-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition of one or several amino acids at the C-terminus does not generally alter the function of the protein. In another preferred embodiment, the C-terminus of the recombinant protein a-mer is added with one amino acid residue Cys.
Variants of the protein include: homologous sequences, conservative variants, allelic variants, natural mutants, proteins encoded by DNA that can hybridize to DNA of recombinant protein A having antibody binding ability under high or low stringency conditions, and proteins or proteins obtained using antisera to the recombinant protein A having antibody binding ability.
As used herein, unless otherwise indicated, the terms "recombinant protein a", "SpA recombinant protein", "recombinant protein a-mer", and "recombinant protein a-mer" are used interchangeably and refer to the recombinant protein of the invention that is obtained by amino acid sequence engineering of wild-type SpA.
The recombinant protein a-mers of the invention can be produced by chemical synthesis, or from prokaryotic or eukaryotic hosts using recombinant techniques. The host can be any cell known to those skilled in the art that can express recombinant proteins (e.g., bacterial, yeast, higher plant, insect, and mammalian cells). In another preferred embodiment, the host is E.coli BL21(DE 3).
In another preferred embodiment, the "Z fragment mutant" of the present invention is: the amino acids (asparagine) at positions 3, 6 and 23 of the Z fragment were changed to aspartic acid, leucine and threonine, respectively, to obtain a Z fragment mutant (shown in SEQ ID NO. 3).
As used herein, the term "linker peptide" refers to a short peptide that is positioned between two mutein fragments and that serves as a linker. The length of the linker peptide is not particularly limited. The length of the linker peptide is typically 5-50 amino acids. In another preferred embodiment, the linker peptide does not or does not significantly affect the formation of the correct fold and spatial conformation of the mutein fragment. In another preferred embodiment, the linker peptide sequence is about 5 to 50 amino acids. In another preferred embodiment, the linker peptide sequence is about 10-20 amino acids. In another preferred embodiment, the linker peptide comprises the sequence shown in SEQ ID NO. 4. In another preferred embodiment, the linker peptide has the sequence shown in SEQ ID NO. 4.
The invention also provides a nucleotide sequence for encoding the recombinant protein A-mer. Accordingly, the present invention includes protein DNA sequences useful for the production of mutants by expression in recombinant hosts by the prior art. (the present invention includes all nucleotide sequences which may encode the recombinant protein A-mer based on codon degeneracy.) in another preferred embodiment, the recombinant protein A-mer comprises a nucleotide sequence as set forth in SEQ ID No. 1. In another preferred embodiment, the recombinant protein A-mer has the nucleotide sequence as shown in SEQ ID No. 1.
The method for synthesizing the full-length nucleotide sequence or the fragment of the recombinant SpA protein of the present invention may be a chemical synthesis method, a PCR amplification method, or a recombinant method. In another preferred embodiment, the nucleotide sequence is obtained by chemical synthesis.
The invention relates to a recombinant expression vector for expressing SpA recombinant protein, which comprises a nucleotide sequence and a vector sequence for coding the SpA recombinant protein. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known to those skilled in the art. In general, any plasmid and vector can be used in the present invention as long as they can stably replicate in a host. In another preferred embodiment, the expression vector comprises one or more selectable marker genes to provide a phenotypic profile for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli. In another preferred embodiment, the expression vector is selected from the group consisting of pGEX or pET series vectors. In another preferred embodiment, the expression vector is the pET30a (+) vector.
The invention also provides a host for expressing the SpA recombinant protein, and the expression host comprises the recombinant expression vector. The host may be any expression cell known to those skilled in the art, and may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. In another preferred embodiment, the host is E.coli BL21(DE 3).
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation may also be by electroporationThe method is carried out. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The obtained transformant can be cultured by a conventional method to express the recombinant SpA protein of the invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. Culturing under conditions suitable for growth of the host cell, and inducing the host cell to express the target protein by a suitable method (e.g., temperature shift or chemical induction) after the host cell has grown to a suitable cell density.
Therefore, the invention also provides a fermentation process of the host expressing the SpA recombinant protein. The process comprises the steps of culturing a certain amount of seed bacteria through multi-stage seeds, adding the seed bacteria into a fermentation culture medium according to a certain inoculation amount, and expressing recombinant protein after inducing the seed bacteria for a certain time through an inducer.
In another preferred embodiment, the inoculation amount of the seed bacteria is 5% -15%. In another preferred embodiment, the inoculation amount of the seed bacteria is 10%. In another preferred embodiment, the inducer is lactose or IPTG. In another preferred embodiment, the inducer is IPTG. In another preferred embodiment, the induction time is 1-5 hr. In another preferred embodiment, the induction time is 2.5 hr.
The SpA recombinant protein can be expressed in cells or cell membranes or secreted out of the cells. If necessary, the protein of interest can be isolated and purified by various separation methods using its physical, chemical and other properties. The above separation and purification methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment by protein precipitation (salting-out method), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.
Applications of
The SpA recombinant protein can be used for preparing an affinity medium for purifying antibodies. Accordingly, the present invention provides an affinity chromatography medium useful for purifying antibodies, comprising an affinity medium, and a recombinant SpA protein of the invention attached to the affinity medium.
The affinity medium is not particularly limited in the present invention, and may be any affinity medium suitable for attachment of protein a, including but not limited to: agarose (beads), sephadex, cellulose, macroporous adsorption resin and other carriers.
The method for attaching the SpA recombinant protein serving as the ligand to the affinity medium in the affinity chromatography process can be a carrier binding method, a physical adsorption method, a crosslinking method or an embedding method. In another preferred embodiment, the method of attaching the protein to the carrier is a cross-linking method. In another preferred embodiment, the protein and carrier are cross-linked together by thioether linkages.
The main advantages of the invention are:
the SpA recombinant protein has good stability under alkaline conditions and strong binding capacity to antibodies or antibody-like proteins, and an affinity chromatography medium prepared from the SpA recombinant protein has high dynamic loading capacity, so that the protein purification process is simplified, and the purification efficiency is improved.
The present invention will be further described with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: conditions as described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,2001), or as recommended by the manufacturer.
Example 1: preparation of recombinant protein A multimers
a. Synthesis of recombinant protein A-mer Gene
A cDNA sequence (shown as SEQ ID No. 1) of a recombinant protein A polymer is synthesized by using a whole gene, the sequence has the full length of 768bp (consigned to Shanghai Czejust bioengineering, Inc for completion), the sequence comprises an initiation codon ATG and a termination codon TAA, and the synthesized sequence is connected into an expression vector pET30a (+), so as to obtain an expression plasmid which is named as pET30 a-4Z' and is shown as figure 1.
b. Sequence identification and expression
With CaCl2Method E.coli BL21(DE3) was transformed with the plasmid pET30 a-4Z', grown overnight on LB plates containing 50. mu.g/mL kanamycin (kanamyin, Kana for short), and then a single colony was picked up and inoculated into LB liquid medium (50. mu.g/mL kana) at 37 ℃ for 8hr with shaking at 200 rpm. Adding 2mL of the bacterial solution into 100mL of LB culture medium (50. mu.g/mL kana), carrying out shaking culture at 37 ℃ and 220rpm until OD600 is 0.6-0.8, adding an inducer isopropyl-beta-D-thiogalactoside (IPTG) to a final concentration of 0.3mM, placing the mixture in a shaking table at 37 ℃ for induction expression, respectively carrying out induction for 2hr and 3hr, then centrifuging at 10000rpm for 10min, collecting thalli, and discarding supernatant. The thalli is added with a buffer solution for heavy suspension (50mM Tris-HCl, pH7.5), high-pressure homogenization and crushing are carried out, centrifugation is carried out at 10000rpm for 30min, and supernatant is collected and is detected by 12% polyacrylamide gel (SDS-PAGE) electrophoresis. Meanwhile, a part of the bacterial liquid is taken and sent to a sample for gene sequencing verification.
As a result, as shown in FIG. 2, the size of the recombinant protein A dimer was consistent with the expected molecular weight. The gene sequencing result is completely consistent with the theory.
The correctly confirmed strains were inoculated into 30mL of LB medium (50. mu.g/mL kana) and cultured (37 ℃,200 rpm). Stopping culturing when OD600 reaches about 0.7-0.8, subpackaging the bacterial liquid into 1mL freezing tubes, and adding glycerol until the final concentration is 15%. Storing at-70 deg.C, and establishing seed bank.
c. Fermentation production
The fermentation seed culture medium is LB culture medium, the batch culture medium of the fermentation tank is peptone, yeast powder, sodium chloride and magnesium sulfate, and the supplementary culture solution of the fermentation tank is glucose, peptone, yeast powder, disodium hydrogen phosphate, sodium dihydrogen phosphate, ammonium sulfate, 10% defoamer, metal ions and vitamins.
The fermentation process comprises the following steps:
first-order seed culture: the seed solution was inoculated into a triangular flask containing 30mL/250mL LB medium (50. mu.g/mL kana) at an inoculum size of 1%, and cultured in a shaker (180rpm/min) at 37 ℃ until the OD600 became 0.2 to 0.3.
Secondary seed culture: the first-order seeds were inoculated into a triangular flask containing 200mL/500mL LB medium (50. mu.g/mL kana) at an inoculum size of 5%, and cultured in a shaker (180rpm/min) at 37 ℃ until the OD600 was 1-1.8.
Adding the second-level seeds into a fermentation tank according to the inoculation amount of 10 percent, starting fermentation production, setting the pH to be 7.0, controlling the temperature to be 37 ℃, and controlling the dissolved oxygen to be more than 20 percent. Feeding materials 2hr after inoculation, measuring the OD600 and glucose content every other hour, adding IPTG with final concentration of 0.3mM when OD600 reaches about 40-50, inducing for 2.5hr, placing in a tank, centrifuging, collecting thallus, and freezing.
d. Purification preparation of protein samples
Adding buffer solution into the thalli for resuspension (50mM Tris-HCl, pH7.5), homogenizing and crushing at high pressure, centrifuging for 30min at 10000rpm, collecting supernatant, filtering the supernatant by using a 0.22 mu M filter membrane, precipitating the hybrid protein at low pH, loading an SP column, performing gradient elution by using 50mM Tris-HCl, pH7.5 (containing 0.5M NaCl) to obtain a purified recombinant protein A polymer, and detecting the purity by using HPLC (high performance liquid chromatography) to be more than 95%. The chromatogram is shown in FIG. 3.
Example 2: load cell determination
The recombinant protein A polymer prepared in example 1 was used as a ligand, and monodisperse porous copolymer polystyrene-divinylbenzene (PS/DVB) microspheres were used as a carrier. Stirring and reacting for 8hr at 25 ℃ in a PB buffer solution with the pH value of 6-8, crosslinking the protein and the microsphere carrier through a thioether bond, washing the obtained medium for several times by using the PB buffer solution, removing unbound protein, and drying in vacuum to obtain an affinity chromatography medium (named as a recombinant protein A polymer medium).
The following affinity chromatography media were loaded using a cell supernatant sample (2.0g/L) expressing human IgG:
a recombinant protein a mer medium;
MabCaptureTMmedia A (available from AB Applied Biosystems, Inc., cat # 4374730) was used as a contrast medium.
The loading buffer was 10mM PB +0.2M NaCl, pH 7.5. The elution buffer was 20mM Sodium Citrate +0.2M NaCl, pH3.7, and the flow rate was 115cm/h, and the samples were sequentially loaded and eluted. And collecting the eluent according to the spectrogram. The protein concentration in the eluate was determined separately by the a280 method, as shown in table 1, wherein the loading was calculated according to formula I.
Wherein V1 is the volume of eluent;
c1 is eluent protein concentration;
vc is the column volume.
TABLE 1
The results are shown in Table 1, and show that the capacity (19.0g/L) of the recombinant protein A polymer medium prepared by the invention is remarkably superior to that of MabCapture ATMLoading (7.9 g/L).
Example 5: recombinant protein A multimers and recombinant wild-type protein A (rPA) alkali resistance assays
10mL of 1M NaOH solution was added to 10mL of the recombinant protein A-mer prepared in example 1 (6mg/mL), mixed (NaOH concentration in the mixture was 0.5M), incubated at 37 ℃ in an incubator, and subjected to electrophoresis at 4hr,8hr,16hr, and 24hr, respectively, to analyze the samples without treatment (0hr) and with alkali treatment by 12% SDS-PAGE, wherein the amount of the sample applied was 5. mu.g at each time point, and the results of the electrophoresis are shown in FIG. 4 (a).
0.6mL of recombinant wild-type protein A (E.coli recombinant expression wild-type staphylococcus aureus protein A, concentration is 1.2mg/mL) solution is added with 5.4mL of 10mM PB, pH is 7.2, the mixture is mixed uniformly, 1mL of sample is remained, an equal volume of 1M NaOH solution is added into the rest solution (5mL), the mixture is mixed uniformly (the concentration of NaOH in the mixture is 0.5M), the mixture is incubated in a 37 ℃ incubator, samples are taken for 4hr,8hr and 16hr respectively, samples are analyzed by 12% SDS-PAGE electrophoresis, samples which are not processed (0hr) and samples which are processed by alkali liquor, the sample loading amount of each time point is 5 mu g, and the electrophoresis result is shown in a graph (b) in a figure 4.
The results show that the recombinant protein A multimer still has protein bands after 16hr of alkaline incubation, whereas the recombinant wild-type protein A is severely degraded after 4hr of alkaline incubation. The recombinant protein A polymer of the invention has stronger alkali resistance.
Example 6: determination of dynamic adsorption capacity of human IgG1 by recombinant protein A polymer medium
The recombinant protein A-mer media prepared in example 1 was subjected to an on-line Cleaning test (CIP) with a Cleaning buffer of 0.5M NaOH, a contact time of 10min per cycle, and a Dynamic Binding Capacity (DBC) was measured every 20 cycles.
The DBC determination method is as follows: the sample was human IgG1 and the flow rate was 1mL/min, and the loading was stopped by 10% breakthrough. Calculating DBC according to equation II10％。
DBC10％＝(V10％-V0)*C/Vc II
Wherein, V10％Sample volume at 10% penetration;
V0the volume of the residual sample in the system pipeline is calculated;
c is the sample concentration;
Vcis the column volume.
The result shows that the DBC of the recombinant protein A polymer medium still remains more than 90 percent after the recombinant protein A polymer medium is subjected to CIP for 120 times in 0.5M NaOH alkali liquor, and the recombinant protein A polymer medium has good alkali resistance.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A recombinant protein A mer, wherein the structure of the recombinant protein A mer is represented by formula (I):
Pa-(Za-L)n-Za-Pb (I)
in the formula (I), the compound is shown in the specification,
pa is absent, Met, a tag sequence for purification, or a combination thereof;
each Za is independently the amino acid sequence of a Z fragment of staphylococcus aureus protein A, and the sequence of Za is shown as SEQ ID No. 3;
l is independently a QKDAVFP amino acid sequence;
n is 3;
pb is nothing, Cys, a purified tag sequence, or a combination thereof.
2. The recombinant protein a-mer of claim 1, further comprising the amino acid residue Cys at the C-terminus of the recombinant protein a-mer.
3. The recombinant protein a-mer of claim 1, wherein the recombinant protein a-mer has an amino acid sequence as set forth in SEQ ID No.: 5.
4. The recombinant protein a mer of claim 1, wherein the recombinant protein a mer has the structure of formula (II):
Met-Za-L-Za-L-Za-L-Za-Cys (II)
wherein Za and L are as defined above.
5. A polynucleotide encoding the recombinant protein A-mer of any one of claims 1-4.
6. A vector comprising the polynucleotide of claim 5.
7. A host cell comprising the vector of claim 6 or a polynucleotide of claim 5 integrated into its genome.
8. A method of producing a recombinant protein a mer of claim 1, comprising the steps of:
(a) culturing the host cell of claim 7 so as to express the recombinant protein a-mer of claim 1; and
(b) isolating or purifying the recombinant protein A-mer from the culture system.
9. An affinity chromatography medium comprising a recombinant protein a-mer of any one of claims 1-4.
10. A purification method, comprising the steps of: for the raw material to be purified, affinity chromatography is carried out using the affinity chromatography medium of claim 9.
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| ES2909833T3 (en) * | 2016-05-11 | 2022-05-10 | Cytiva Bioprocess R & D Ab | Cleaning and/or disinfection method of a separation matrix |
| GB201904125D0 (en) | 2019-03-26 | 2019-05-08 | Ge Healthcare Bio Sciences Ab | Method for sanitization of a chromatography column |
| CN111057153B (en) * | 2019-12-06 | 2021-09-07 | 广州康盛生物科技股份有限公司 | Immunoglobulin binding protein and preparation method and application thereof |
| GB202113626D0 (en) | 2021-09-24 | 2021-11-10 | Cytiva Bioprocess R & D Ab | FC binding polypeptides |
| GB202203478D0 (en) | 2022-03-14 | 2022-04-27 | Cytiva Bioprocess R & D Ab | Vh3 binding polypeptides |
| CN114409749A (en) * | 2022-03-15 | 2022-04-29 | 青岛科诺赛生物科技有限公司 | High-stability recombinant protein A with antibody binding force |
| CN117327159A (en) * | 2023-07-31 | 2024-01-02 | 杭州纽龙生物科技有限公司 | Alkali-resistant mutation method for protein A |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1642976A (en) * | 2002-03-25 | 2005-07-20 | 阿默森生物科学有限公司 | A mutated immunoglobulin-binding protein |
| CN103214563A (en) * | 2013-03-26 | 2013-07-24 | 江南大学 | Performance improved recombination staphylococcus aureus protein A affinity ligand and construction method thereof |
| WO2013147691A1 (en) * | 2012-03-28 | 2013-10-03 | Ge Healthcare Bio-Sciences Ab | Affinity chromatography matrix |
| CN103403020A (en) * | 2010-12-21 | 2013-11-20 | Jsr株式会社 | Novel alkali-resistant variants of protein a and their use in affinity chromatography |
-
2014
- 2014-09-16 CN CN201410471277.2A patent/CN105481954B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1642976A (en) * | 2002-03-25 | 2005-07-20 | 阿默森生物科学有限公司 | A mutated immunoglobulin-binding protein |
| CN103403020A (en) * | 2010-12-21 | 2013-11-20 | Jsr株式会社 | Novel alkali-resistant variants of protein a and their use in affinity chromatography |
| WO2013147691A1 (en) * | 2012-03-28 | 2013-10-03 | Ge Healthcare Bio-Sciences Ab | Affinity chromatography matrix |
| CN103214563A (en) * | 2013-03-26 | 2013-07-24 | 江南大学 | Performance improved recombination staphylococcus aureus protein A affinity ligand and construction method thereof |
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