CN103214563B - A kind of recombination staphylococcus aureus albumin A affinity ligand of improvement in performance and construction process thereof - Google Patents
A kind of recombination staphylococcus aureus albumin A affinity ligand of improvement in performance and construction process thereof Download PDFInfo
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Abstract
The invention discloses a kind of recombination staphylococcus aureus albumin A affinity ligand and construction process thereof of improvement in performance.The present invention utilizes molecular biology method, the B sequence choosing native protein A carries out molecular modification, interpolation 2 halfcystines are held at the C of B sequence, thus can by the chromatography substrate of albumin A by the coupling of dibit point, make connection more stable, after B sequence second Loop, with the addition of 6 glycine increase its length, reduce the bonding force between antibody, thus make elution requirement gentleer.On this basis the performance of albumin A resisting high-concentration alkali is transformed, the l-asparagine of B sequence the 23rd, 30 and phenylalanine are replaced to Threonine and L-Ala respectively, obtains the Z sequence that alkaline resistance properties is higher.Then, utilize isocaudarner by the series connection of the Z sequence of different quantities head and the tail, and utilize colibacillary efficient expression system to carry out overexpression.The recombinant protein A of expression is coupled to agarose matrix and prepares affinity chromatography filler, and for antibody purification, result shows that recombinant protein A affinity ligand prepared by the present invention has good elution property and alkaline resistance properties.
Description
Technical field
The invention belongs to genetically engineered field, relate to a kind of recombination staphylococcus aureus albumin A affinity ligand and construction process thereof of improvement in performance.
Background technology
Staphylococcus aureus protein A is called for short albumin A, is a kind of protein of staphylococcus aureus surface, is connected to muramyl peptide glycan by covalent linkage.There is very high E, D, A, B, C sequence of five homologys in Protein A molecules, because the Fc section of energy and people and multiple Mammals IgG combines, be therefore referred to as antigen-binding site.Albumin A is combined with the Fc section of antibody does not affect the adsorption activity of antibody to antigen, is therefore widely used in the affinity purification of antibody.Because the bonding force of B structural domain antagonist is the most stable, and this body structure is also the most stable, and therefore people are to the most study of B sequence.B sequence is made up of with two Loop being connected spiral fragment 3 α spirals, is Helix1-Loop1-Helix2-Loop2-Helix3 successively according to the order of holding C to hold from N.
When taking albumin A as affinity ligand absorption IgG, bonding force is very strong, needs to use lower pH elutriant (about pH3.0), often affects the activity of IgG.Protein A affinity chromatography post in use can remain the impurity such as Nucleotide, denatured protein, lipid and microorganism in post, needs to use incumbent firms to remove, and wherein NaOH is scavenging solution comparatively cheap and conventional in industrial production.Although native protein A has good tolerance to alkaline environment, still can not meet life-time service highly basic and it is cleaned.Although E, D, A, B, C five structural domains of native protein A antigen-binding site have very high homology each other, they there are differences the binding ability of IgG, cause the suitableeest elution requirement different.
Therefore, the present invention is directed to above some, choose B structural domain and molecular modification carried out to native protein A.Molecular biology method is utilized to carry out transforming and being connected from nucleotide sequence, escherichia coli high-level expression system is utilized to realize high expression, obtain a series of transformation sequences to be composed in series and C holds the recombinant protein A of an interpolation halfcystine, the recombinant protein A obtained is prepared affinity chromatography filler as affinity ligand, for the separation and purification of antibody, possess gentleer elution property and higher alkaline resistance properties.
Summary of the invention
The object of this invention is to provide a kind of recombination staphylococcus aureus albumin A affinity ligand of improvement in performance, for affinity chromatography filler prepared by antibody, there is good elution property and alkaline resistance properties.
Described recombinant protein A affinity ligand is by 1 to 10 Z sequences a series of protein in series.Z sequence is that B sequence is through improved product, and the difference of B sequence is to add 6 glycine between Loop2 and Helix3, and the l-asparagine of the 23rd, 30 and phenylalanine are replaced to Threonine and L-Ala respectively, in order to realize albumin A and the more stable connection of filler matrix, with the addition of 2 halfcystines at the C end of B protein sequence, thus realize realizing dibit point with matrix and being connected.Be z by the unnamed gene of expressing Z sequence, utilize isocaudarner to be connected by the z of 1 to 10 different quantitiess, in expressed recombinant protein A, every two Z sequences are connected by two Serines.
The step of the construction process of the recombinant protein A affinity ligand of described improvement in performance is:
(1) design of primers
The B fragment of the present invention to albumin A carries out molecular modification, according to over-lap PCR and isocaudarner catenation principle design primer, by improved B fragment called after Z, reforming content is the transformation of Z (6G), Z (N23T, F30A) transformation, C holds the interpolation of halfcystine and the splicing of 1 to 10 z nucleotide sequences, wherein the transformation of Z (6G) after B sequence second Loop, adds 6 glycine increase Loop2 length, thus reduce the bonding force intensity of itself and antibody, wash-out is more easily carried out, Z (N23T, F30A) transformation is the to B sequence the 23rd, the l-asparagine of 30 and phenylalanine carry out rite-directed mutagenesis, replace with Threonine and L-Ala respectively, reduce the desamidation of B sequence, strengthen the tolerance performance to high-concentration alkali liquor, C holds the interpolation of halfcystine to be hold interpolation 3 halfcystines at the C of B sequence, realize dibit point be connected to realize recombinant protein A affinity ligand and agarose plugs matrix, Nhe I is added in primer, Xba I, Noc I and BamH I tetra-restriction enzyme sites, isocaudarner action principle is utilized to be joined end to end by the z nucleotide sequence of different number, the recombinant protein A expressing gene of composition containing different number z sequence series connection,
(2) structure of recombinant bacterium
The primer of the present invention designed by step (1), with staphylococcus aureus gene group for template carries out over-lap PCR, the object nucleotide sequence z needed for acquisition, utilizes carrier T pMD18-T construction recombination plasmid pMD18-T-z
1, transformed competence colibacillus E.coliJM109 carries out the preservation of glycerine frozen pipe, carries out different number z sequence (z based on this
n) structure, then utilize the z that Noc I and BamH I two restriction enzyme sites will build
nsequence and expression plasmid pET-28a carry out enzyme and cut and be connected, and obtain recombinant expression plasmid pET-28a-z
n, last transformed competence colibacillus E.coli BL21 (DE3), obtains expression strain E.coliBL21 (the DE3)/pET-28a-z of recombinant protein A
n, prepare the preservation of glycerine frozen pipe for subsequent use;
(3) expression of recombinant protein A
The present invention utilizes IPTG to recombinant bacterium E.coli BL21 (the DE3)/pET-28a-z constructed by step (2)
ncarry out abduction delivering, obtain the recombinant protein A with 1-10 Z fragment series connection, with Z
nrepresent the recombinant protein A of the individual Z fragment series connection of n (n is 1-10).
The present invention utilizes the B sequence of molecular biology method to native protein A to carry out molecular modification, first the B sequence of albumin A is carried out simultaneously to the transformation of elution property and alkaline resistance properties, and realize improved 1 to 10 z closely to connect, build recombinant expression plasmid pET-28a-z
nand transformed competence colibacillus E.coli BL21 (DE3) expresses.
The recombinant protein A utilizing the present invention to express is prepared affinity chromatography filler antagonist as affinity ligand and is had good binding ability, elution property and alkaline resistance properties: use pH4.0,5.0 and 6.0 elutriant can realize respectively 97.7%, 89.4% and 67.5% eluting rate; Use the NaOH solution of 2.0mol/L to soak 7h and 14h, bovine coloctrum IgG combination rate and be respectively 86.1% and 72.8%.
Key of the present invention is: carry out elution property transformation to B sequence, adds 6 glycine, make elution requirement gentleer between B sequence Loop2 and Helix3; To the transformation of B sequence alkaline resistance properties, utilize overlapping pcr that the l-asparagine of the 23rd, 30 and phenylalanine are replaced with Threonine and L-Ala respectively, reduce desamidation, enhancement sequences is to the tolerance performance of high concentration alkali solution; Utilize isocaudarner action principle, different number z sequence is connected, obtain a series of recombinant protein A comprising different number Z fragment through conversion and abduction delivering.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE proof diagram that the recombination staphylococcus aureus albumin A of a kind of improvement in performance of the present invention is expressed.1，marker；2，E.coli BL21(DE3)/pET28a-z
5；3，E.coli BL21(DE3)/pET28a。
Fig. 2 is the procedure chart of recombination staphylococcus aureus protein A affinity chromatography filler purifying bovine coloctrum IgG prepared by the present invention.
Fig. 3 is the SDS-PAGE proof diagram of recombination staphylococcus aureus protein A affinity chromatography filler purifying bovine coloctrum IgG prepared by the present invention.1, marker; 2, elutriant; 3, penetrate liquid; 4, crude samples IgG.
Fig. 4 is the elution property analysis of recombination staphylococcus aureus albumin A affinity ligand prepared by the present invention.
Fig. 5 is the alkaline resistance properties analysis of recombination staphylococcus aureus albumin A affinity ligand prepared by the present invention.
Embodiment
The invention will be further described by the following examples:
(1) design of primers
Embodiment 1
Use the B fragment of overlapping pcr to albumin A to transform, and by improved B fragment called after Z fragment, its expressing gene is z.According to the gene order that GeneBank provides, molecular biosciences software DNAMAN is utilized to design the forward primer F of z sequence
0with reverse primer R
0:
F
0：
CGGATAACAAATTCAAC
R
0：
TTA
GCAACA TTTTGGTGCTTGTGC。
Forward primer and reverse primer introduce Noc I and BamH I restriction enzyme site respectively, and wherein italicized item is restriction enzyme site, and underscore part is the codon of two halfcystines added.
After B fragment second Loop, add 6 glycine, reduce the bonding force intensity of albumin A and IgG, be expressed as Z (6G); In order to increase the alkaline resistance properties of albumin A, the phenylalanine of the l-asparagine of the 23rd and the 30th being replaced to Threonine and L-Ala respectively, represents with Z (N23T, F30A).Gene order design z gene order according to GeneBank provides:
gCGGATAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTACATTT ACCTAACTTA
aCCgAAGAACAACGCAATGGT
gCCaTCCAAAGCTTAAAAGATGACCCAAGCCAA
gGTGGCGGAGGGGGCGGTaGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAA
tGTTGCtAA
wherein underscore mark is genetic modification part, and italic mark part is restriction enzyme site, adds Nhe I and Noc I two restriction enzyme sites in z front end, XbaI and BamH I two restriction enzyme sites are added in rear end.According to over-lap PCR principle, molecular biosciences software DNAMAN is utilized to design two pairs of primers:
F
1：ATGACCCAAGCCAAGGTGGCGGAGGGGGCGGTAGCGCTAACCTTTTA
R
1：TAAAAGGTTAGCGCTACCGCCCCCTCCGCCACCTTGGCTTGGGTC
F
2：ACTTAACCGAAGAACAACGCAATGGTGCCATCCAAAGCTTAA
R
2：TTGGATGGCACCATTGCGTTGTTCTTCGGTTAAGT
Wherein F
1and R
1the forward and reverse primer of Z (6G) respectively, F
2and R
2the forward and reverse primer of Z (N23T, F30A) respectively.Sangon Biotech (Shanghai) Co., Ltd. is transferred to synthesize designed primer.
(2) recombinant vectors pMD18-T-z
1structure
Embodiment 2
Extract L-form staphylococcus aureus, utilize the F that embodiment 1 designs
0, R
0for primer, L-form staphylococcus aureus is template, utilizes Pfu archaeal dna polymerase to carry out PCR to B sequence.According to Pfu archaeal dna polymerase specification sheets, 50 μ L are selected to carry out.
PCR reaction conditions: denaturation 94 DEG C reaction 5min; Sex change 94 DEG C reaction 40s, annealing temperature 56 DEG C reaction 1min30s, elongating temperature 72 DEG C extends 30s, carries out 30 circulations; Last 72 DEG C keep 10min.
Utilize sepharose to reclaim test kit and reclaim B fragment.Use the method for over-lap PCR, utilize Pfu DNA exo+ polymerase, with the B fragment be recovered to for template, respectively with the F that embodiment 1 designs
0and R
1, F
1and R
0for primer carries out PCR, reaction system and condition the same.PCR is reclaimed product to mix, using this mixture as template, with F according to volume ratio 1: 1
0and R
0for template carries out PCR, reaction system and condition the same, sepharose reclaims the sequence of increase by 6 codon glycines.With system, alkali resistance is suddenlyd change in the same way again, respectively with the F that embodiment 1 designs
0and R
2, F
2and R
0and F
0and R
0for primer, with the improved sequence obtained for template carries out over-lap PCR, glue reclaims and obtains z sequence.With F
0and R
0for primer, z sequence are template, utilize Ex-Taq archaeal dna polymerase to carry out PCR, reaction system and condition the same, obtain two ends respectively with the z sequence of a VITAMIN B4, utilize base pair complementarity principle to be connected on carrier T pMD18-T by z sequence, obtain recombinant plasmid pMD18-T-z
1.By recombinant plasmid transformed competence colibacillus E.coli JM109, obtain recombinant bacterium E.coliJM109/pMD18-T-z
1carry out the preservation of glycerine frozen pipe for subsequent use.
(3) recombinant vectors pET-28a-z
nstructure
Embodiment 3
Z
nrepresent that n (n is 1-10) individual z sequence joins end to end.Using Nhe I and BamHI as first group of restriction restriction endonuclease, XbaI and BamH I is as second group of restriction restriction endonuclease.The recombinant bacterium E.coliJM109/pMD18-T-z that incubated overnight embodiment 2 obtains
1, plasmid extraction pMD18-T-z
1, with two groups of enzymes respectively to recombinant plasmid pMD18-T-z
1carry out endonuclease reaction.Glue reclaims two groups of digestion products, utilizes T4DNA ligase enzyme by two groups of products in 16 DEG C of connections of spending the night, and obtains containing two end to end recombinant plasmid pMD18-T-z of z sequence
2, by pMD18-T-z
2the competence E.coli JM109 that is transformed into preserve.
Embodiment 4
The recombinant bacterium E.coli JM109/pMD18-T-z that incubated overnight embodiment 3 obtains
2, extract plasmid pMD18-T-z
2, utilize second group of restriction enzyme of embodiment 3 to pMD18-T-z
1carry out enzyme to cut, first group of restriction enzyme is to pMD18-T-z
2carry out enzyme to cut, carry out connection after being reclaimed by the digestion products of two groups of gained and obtain containing 3 end to end recombinant plasmid pMD18-T-z of z sequence
3, transformed competence colibacillus E.coli JM109 preserves.According to identical method, the two groups of enzymes utilizing embodiment 3 to mention are respectively to pMD18-T-z
2carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 4 end to end recombinant plasmid pMD18-T-z of z sequence
4, transformed competence colibacillus E.coli JM109 preserves; Utilize one group of enzyme of embodiment 3 to pMD18-T-z
2carry out enzyme to cut, another group enzyme is to pMD18-T-z
3carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 5 end to end recombinant plasmid pMD18-T-z of z sequence
5, transformed competence colibacillus E.coli JM109 preserves; The two groups of enzymes utilizing embodiment 3 to mention are respectively to pMD18-T-z
3carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 6 end to end recombinant plasmid pMD18-T-z of z sequence
6, transformed competence colibacillus E.coli JM109 preserves; Utilize one group of enzyme of embodiment 3 to pMD18-T-z
3carry out enzyme to cut, another group enzyme is to pMD18-T-z
4carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 7 end to end recombinant plasmid pMD18-T-z of z sequence
7, transformed competence colibacillus E.coli JM109 preserves; The two groups of enzymes utilizing embodiment 3 to mention are respectively to pMD18-T-z
4carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 8 end to end recombinant plasmid pMD18-T-z of z sequence
8, transformed competence colibacillus E.coli JM109 preserves; Utilize one group of enzyme of embodiment 3 to pMD18-T-z
4carry out enzyme to cut, another group enzyme is to pMD18-T-z
5carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 9 end to end recombinant plasmid pMD18-T-z of z sequence
9, transformed competence colibacillus E.coli JM109 preserves; The two groups of enzymes utilizing embodiment 3 to mention are respectively to pMD18-T-z
5carry out enzyme to cut, the digestion products of two groups of gained is connected, can obtain containing 10 end to end recombinant plasmid pMD18-T-z of z sequence
10, transformed competence colibacillus E.coli JM109 preserves.
Embodiment 5
Incubated overnight Laboratories Accession bacterial strain E.coli JM109/pET-28a and embodiment 4 gained 10 strain recombinant bacterium E.coliJM109/pMD18-T-z
1-10, extract plasmid, utilize restriction enzyme Noc I and BamH I to plasmid pET-28a and pMD18-T-z
1-10carrying out enzyme to cut, both obtaining recombinant plasmid pET-28a-z by carrying out connecting after the recovery of digestion products glue
1-10, transformed competence colibacillus E.coli BL21 (DE3), obtains genetic engineering bacterium E./oliBL21 (DE3)/pET-28a-z that different fragments length recombinant protein A is expressed in 10 strains
1-10, the preservation of glycerine frozen pipe.
Embodiment 6
Activating Sepharose4FF with glycidyl allyl ether and NHS and modify, preservation is for subsequent use.By embodiment 5 gained recombinant bacterium E.coli BL21 (DE3)/pET-28a-z
5carry out ultrasonication after incubated overnight, utilize the Z expressed by IgG affinity chromatography filler purifying
5protein fragments, carries out lyophilize by purified product for subsequent use.By the Z of preparation
5protein fragments is made into proper concn solution, is coupled to modified Sepharose4FF and prepares recombinant protein A affinity chromatography filler.
Embodiment 7
Based on
purifier chromatographic system, recombinant protein A affinity chromatography filler embodiment 6 prepared dress post carries out performance test.Prepare the 0.1mol/L citrate-phosphate disodium hydrogen damping fluid of different pH as elutriant, detect the elution property of filler.Use pH4.0,5.0 and 6.0 elutriant carry out wash-out, achieve the eluting rate of 97.7%, 89.4% and 67.5% respectively.After using the NaOH solution of 2.0mol/L to soak 7h and 14h, the combination rate of affine filler to IgG prepared by recombinant protein A of the present invention is respectively 86.1% and 72.8%.
Claims (6)
1. the recombination staphylococcus aureus albumin A affinity ligand of an improvement in performance, it is characterized in that: described recombination staphylococcus aureus albumin A affinity ligand is by 1 to 10 Z sequences a series of protein in series, Z sequence is that B sequence is through improved product, and the difference of B sequence is to add 6 glycine between Loop2 and Helix3, and by the 23rd, the l-asparagine of 30 and phenylalanine replace to Threonine and L-Ala respectively, in order to realize albumin A and the more stable connection of filler matrix, with the addition of 2 halfcystines at the C end of B protein sequence, thus realize realizing dibit point with matrix and being connected, be z by the unnamed gene of expressing Z sequence, isocaudarner is utilized to be connected by the z of 1 to 10 different quantitiess, in expressed recombinant protein A, every two Z sequences are connected by two Serines,
Wherein, described transformation, its reforming content is the transformation of Z (6G), Z (N23T, F30A) transformation, C holds the interpolation of halfcystine and the splicing of 1 to 10 z nucleotide sequences, wherein the transformation of Z (6G) after described B sequence second Loop, adds 6 glycine increase Loop2 length, thus reduce the bonding force intensity of itself and antibody, wash-out is more easily carried out, Z (N23T, F30A) transformation is the to B sequence the 23rd, the l-asparagine of 30 and phenylalanine carry out rite-directed mutagenesis, replace with Threonine and L-Ala respectively, reduce the desamidation of B sequence, strengthen the tolerance performance to high-concentration alkali liquor, C holds the interpolation of halfcystine to be hold interpolation 2 halfcystines at the C of B sequence, realize dibit point be connected to realize recombinant protein A affinity ligand and agarose plugs matrix, Nhe I is added in primer, Xba I, Noc I and BamH I tetra-restriction enzyme sites, isocaudarner action principle is utilized to be joined end to end by the z nucleotide sequence of different number, the recombinant protein A expressing gene of composition containing different number z sequence series connection, the gene order of described Z sequence is as shown in sequence table SEQ ID.3.
2. one according to claim 1 transformable recombination staphylococcus aureus albumin A affinity ligand, is characterized in that: the aminoacid sequence of described Z sequence is as shown in sequence table SEQ ID No.8.
3. one according to claim 1 transformable recombination staphylococcus aureus albumin A affinity ligand, is characterized in that: the aminoacid sequence after 1 to 10 Z sequence series connection is respectively as shown in sequence table SEQ ID No.8-17.
4. a construction process for property as claimed in claim 1 transformable recombination staphylococcus aureus albumin A affinity ligand, is characterized in that: clone and transform 3 pairs of primers F of Z gene order
0and R
0, F
1and R
1, F
2and R
2its nucleotide sequence is respectively SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, utilize over-lap PCR to obtain z sequence, use one group of isocaudarner and restriction enzyme BamH I that 1 to 10 z sequences are carried out series connection and obtain z
nsequence, utilizes one group of restriction enzyme by z
nbe connected to plasmid pET-28a, transformed competence colibacillus E.coli BL21 (DE3) obtains the recombinant protein A bacterial strain producing the series connection of different number z sequence, obtains by the IPTG induction of certain final concentration the recombination staphylococcus aureus albumin A affinity ligand be made up of 1-10 z sequence.
5. construction process according to claim 4, is characterized in that: described isocaudarner is Nhe I and Xba I.
6. construction process according to claim 4, is characterized in that: described restriction enzyme is Noc I and BamH I.
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| CN104513309A (en) * | 2013-09-26 | 2015-04-15 | 上海亨臻实业有限公司 | Protein function structural domain hinge peptide and applications thereof |
| CN105481954B (en) * | 2014-09-16 | 2021-03-26 | 亿一生物制药(北京)有限公司 | Recombinant protein A and application thereof |
| CN106117324A (en) * | 2016-07-29 | 2016-11-16 | 湖北爱晟生物科技有限公司 | Recombinant protein A and encoding gene thereof and application |
| CN106317227B (en) * | 2016-08-29 | 2019-04-12 | 江南大学 | Affinity ligand, construction method, affinity chromatography medium, preparation method and application |
| CN109721645A (en) * | 2017-12-29 | 2019-05-07 | 兆生生物科技南京有限公司 | A kind of albumin A of gene mutation and its application |
| CN114605508B (en) * | 2022-05-11 | 2022-07-29 | 北京达成生物科技有限公司 | Antibody binding proteins capable of binding to the Fc region of an antibody molecule and uses thereof |
| CN116836245B (en) * | 2023-06-01 | 2024-01-26 | 苏州华诺生物科技有限公司 | Recombinant protein synthesized based on characteristic algorithm control and application thereof |
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