CN109384852A - Recombinate preparation, characterization and the application of Martentoxin - Google Patents
Recombinate preparation, characterization and the application of Martentoxin Download PDFInfo
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Abstract
The present invention relates to the preparations and characterization of recombination Martentoxin.Specifically, Martentoxin gene connect to obtain recombinant expression carrier pETDuent-1-MarTX with carrier pETDuent-1 by the present invention, which is converted to E.coli Origami B (DE3), screening obtains efficient expression strain.By inducing expression, affinitive layer purification and characterization, the correct polypeptide of various aspects physicochemical property-recombination MarTX toxin is obtained.It is found through electro physiology measuring, recombination MarTX toxin provided by the invention has bioactivity, can significantly inhibit potassium ion (BK) channel current of big conductance calcium ion and voltage activation.The present invention realizes the green manufacturing of MarTX toxin, and the MarTX Output of toxin of acquisition is high, with high purity, and physiological activity with higher.
Description
Technical field
The present invention relates to preparation, characterization and the applications of recombination Martentoxin, belong to polypeptide toxin and biotechnology engineering
Field.In particular it relates to it is a kind of separated from buthus martensii Karscs (Buthus martensi Karsch, BmK) venom it is pure
Change characterization and the recombinant toxin that obtained toxin-Martentoxin is prepared by recombinant technology and physicochemical property in big electricity
Lead the application in channel potassium ion (BK) of calcium ion and voltage activation.
Background technique
Studies have shown that containing largely with medicine in the venom of hazard biologicals such as snake, centipede, scorpion, spider, sea anemone, cone spiral shell
Manage active polypeptide.There are many venom derivatives containing intramolecular disulfide bond at present is ratified by FDA, treats hypertension, pain
And diabetes [Venoms to drugs:venom as a source for the development of human
therapeutics[M].City:Royal Society of Chemistry,2015].More lps molecules are in clinic
In research, and the diseases such as their targeting epilepsy, cancer and autoimmunities [Curr.Pharm.Des.2007,13,2927-2934；
Toxins 2010,2,2851-2871；Expert Opi.Biol.Ther.2011,11,1469-1484].
Rich in a variety of more with natural activity in the scorpion venom of buthus martensii Karscs (Buthus martensi Karsch, BmK)
Peptide, wherein most may act on ion channel.So far, which has obtained 14 kinds short chain potassium ion by isolating and purifying
Channel blocker (BmTX1-3, BmKTX, BmP01-3, BmP05, BmP09, BmKK1-4 and Martentoxin)
[Biochemistry 1997,36,13473-13482；J.Biol.Chem.2004,279,34562-34569；
Eur.J.Biochem.1997,245,457-64].This Martentoxin (abbreviation MarTX) is by 37 Amino acid profiles, tool
Have three pairs of disulfide bond, be a kind of specific effect in the channel BK short chain polypeptides toxin [J.Neurochem.2003,84,325-
335], nervous system type (alpha+beta 4) BK channel current can be significantly inhibited.
Currently, manually there are mainly three types of the methods of acquisition polypeptide: recombinant expression method, endogenous extraction method and chemical synthesis.
Wherein, endogenous extraction method is most ancient also most straightforward approach, but the polypeptide classes extracted are limited, and yield is lower.Using most
More chemical synthesis is Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase synthesis.This method is related to the synthesis of linear peptide chain, iodine oxidation
System, the protection of cysteine and many organic systems such as the removing of protecting group and refolding strategy, complex steps are complicated, two sulphur
Key fallibility is matched, low yield, and is not able to achieve the green manufacturing of polypeptide drug.And for recombinantly expressing method, due to MarTX poison
Plain molecular weight is smaller, and easily leads to inclusion body expression containing multipair disulfide bond, therefore a large amount of, solvable with the more difficult acquisition of this method
, high purity protein.
Therefore, there is an urgent need in the art to develop a kind of preparation method of MarTX toxin, with obtain high yield, high-purity and
The MarTX toxin biological activity protein of high activity.
Summary of the invention
It is an object of the invention to provide a kind of preparation methods of the MarTX toxin of high yield, high-purity and high activity.
In the first aspect of the present invention, a kind of fusion protein is provided, the fusion protein has Formulas I institute from N-terminal to C-terminal
Show structure:
P1-P2-P3-P4 (Formulas I)
In formula,
P1 is purification tag element；
P2 is maltose-binding protein element (MBP)；
P3 is protease site element；
P4 is MarTX polypeptide element；
"-" indicates the peptide bond of connection said elements.
In another preferred example, the P1 is 6 × His sequence.
In another preferred example, the fusion protein may not include P1 element.
In another preferred example, the sequence of the P2 is SEQ ID NO:4.
In another preferred example, the sequence of the P2 is that one or more amino are carried out on the basis of SEQ ID NO:4
Replacement, missing, change, insertion or the increase of acid, obtained amino acid sequence.
In another preferred example, the protease is selected from the group: HRV HRV 3CP, Thrombin enzyme, Factor Xa,
Enterokinase (Enterkinase), TEV enzyme, Prescission, TAGZyme, SUMO protease, or combinations thereof.
In another preferred example, the protease is Thrombin enzyme.
In another preferred example, the sequence of the P4 is SEQ ID NO:2.
In another preferred example, the sequence of the P4 is that one or more amino are carried out on the basis of SEQ ID NO:2
Replacement, missing, change, insertion or the increase of acid, obtained amino acid sequence.
In another preferred example, the N-terminal of the sequence of the Formulas I or C-terminal can add 1 to 30 amino acid residue, and preferably 1
To 10 amino acid residues, more preferably 1 to 5 amino acid residue.
In another preferred example, between the P1 and P2, between P2 and P3 or between P3 and P4,1 to 30 ammonia can be added
Base acid residue, preferably 1 to 10 amino acid residue, more preferably 1 to 5 amino acid residue.
In another preferred example, the sequence in the sequence of the fusion protein, from N-terminal to C-terminal between each element further include:
P2-P1-P3-P4, P4-P3-P2-P1, P2-P3-P4-P1 or P4-P3-P1-P2.
In the second aspect of the present invention, a kind of isolated polynucleotides are provided, the polynucleotide encoding present invention
Fusion protein described in one side.
In another preferred example, the polynucleotides include the nucleic acid constructs of from 5 ' to 3 ' Formula II,
Z1-Z2-Z3-Z4 (Formula II)
In formula, Z1, Z2, Z3 and Z4 are separately encoded in first aspect present invention P1, P2, P3 and P4 in Formulas I；
Wherein, the sequence of the Z2 is SEQ ID NO:3.
In another preferred example, the sequence of the Z4 is SEQ ID NO:1.
In another preferred example, the sequence in the sequence of the polynucleotides, from 5 ' to 3 ' each elements further include:
Z2-Z1-Z3-Z4, Z4-Z3-Z2-Z1, Z2-Z3-Z4-Z1 or Z4-Z3-Z1-Z2.
In the third aspect of the present invention, a kind of carrier is provided, the carrier contains more described in second aspect of the present invention
Nucleotide.
In another preferred example, the carrier is pETDuet-MarTX-1, and the pETDuet-MarTX-1 is in carrier
In pETDuet-1 obtained by insetion sequence SEQ ID NO:3.
In the fourth aspect of the present invention, a kind of host cell is provided, the host cell contains third aspect present invention
Polynucleotides described in second aspect of the present invention are integrated in the carrier or genome.
In another preferred example, the host cell is E.coli Origami B (DE3).
In fifth aspect present invention, a kind of method for generating fusion protein described in first aspect present invention, packet are provided
Include step:
Under conditions suitable for the expression, host cell described in fourth aspect present invention is cultivated, to give expression to the present invention
Fusion protein described in first aspect.
In sixth aspect present invention, a kind of method for preparing MarTX polypeptide is provided, comprising steps of
(i) the fusion protein digestion described to the first aspect of the present invention with protease, so that digestion products are obtained, the digestion
Product corresponds to MarTX polypeptide；With
(ii) isolated or purified goes out the MarTX polypeptide from digestion products.
In another preferred example, the protease is selected from the group: HRV HRV 3CP, Thrombin enzyme, Factor Xa,
Enterokinase (Enterkinase), TEV enzyme, Prescission, TAGZyme, SUMO protease, or combinations thereof.
In another preferred example, the protease is Thrombin enzyme.
In another preferred example, the MarTX polypeptide that the method purifies is from every liter of resulting yield of expression thallus
>=2mg, preferably >=3mg, more preferably >=4mg.
In seventh aspect present invention, the drug screening method of one kind (alpha+beta 4) BK channel current inhibitor is provided, including
Step:
(i) using method described in sixth aspect present invention, purifying preparation MarTX polypeptide；
(ii) using MarTX polypeptide as positive control, (alpha+beta 4) BK channel current inhibitor drug candidate is screened.
In another preferred example, in the step (ii), the half effect of MarTX polypeptide blocks (alpha+beta 4) BK channel current
Concentration (EC50) value is C0, and the inhibitor drug candidate blocks half effective concentration (EC50) value of (alpha+beta 4) BK channel current
For C1,
As C1≤C0, then the inhibitor drug candidate is an advantage over (alpha+beta 4) BK channel inhibitor of MarTX polypeptide.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the physical map and multiple cloning sites of expression vector pETDuet-1.
Fig. 2 shows MarTX gene PCR process and agarose gel electrophoresis.(A) PCR primer.Upstream primer (Primer-
UP the Xho I site of BamH I site and downstream primer (Primer-DO)) is marked with horizontal line.(B) PCR reaction system group
At.(C) PCR response procedures.(D) agarose gel electrophoresis figure after the reaction was completed.
Fig. 3 shows the double digestion agarose gel electrophoresis figure of expression vector pETDuet-1 and Martentoxin gene.
M indicates DNA marker (DL5000) in figure, is from bottom to up 100bp, 250bp, 500bp, 750bp, 1000bp,
1500bp, 2000bp, 3000bp, 5000bp.Swimming lane 1 is that pETDuet-1 carrier is bis- through restriction enzyme EcoR I and Not I
Digestion is as a result, swimming lane 2 is EcoR I and Not the I double digestion result of Martentoxin genetic fragment.
Fig. 4 shows recombinant expression carrier pETDuet-1-MarTX map schematic diagram.
Fig. 5 shows pGEX-4T-3-MarTX recombinant plasmid and purifying gel chromatography figure: (A) pGEX-4T-3-MarTX table
Up to plasmid schematic diagram；(B) 75 peak figure of MarTX Superdex.
Fig. 6 shows pGEX-6P-1-MarTX recombinant plasmid and purifying gel chromatography figure.(A) pGEX-6P-1-MarTX table
Up to plasmid schematic diagram.(B) 75 peak figure of MarTX Superdex.
Fig. 7 shows pSMT3-MarTX recombinant plasmid and purifying gel chromatography figure.(A) pSMT3-MarTX expression plasmid shows
It is intended to.(B) 75 peak figure of MarTX Superdex.
Fig. 8 shows the purifying of pETDuet-1-MarTX expression of recombinant plasmid toxin.
(A) ni-sepharose purification result.Fusion protein is marked with red line.(B) Amylose Resin column chromatography after digestion
As a result.(C) 75 UV absorption peak figure of gel chromatographic columns Superdex.(D) the SDS-PAGE detection of recombination MarTX after purification.
Fig. 9 shows the one-dimensional hydrogen spectrum of recombination MarTX toxin.
Figure 10 shows the circular dichroism spectra of recombination MarTX toxin.
Figure 11 shows the MALDI-TOF Mass Spectrometric Identification of recombination MarTX toxin.
Figure 12 shows 20 optimal solution structure stacking charts (secondary structure partial stack) of recombination MarTX toxin.It is more
The N-terminal of peptide, C-terminal have marked.
Figure 13 shows the ramachandran map Ramachandran of recombination 20 optimal comformation in solutions of MarTX toxin.
Figure 14 shows the folded structures figure of recombination MarTX toxin and natural toxin.
Red indicates recombination MarTX, and green indicates reported MarTX structure (PDB:1M2S).Disulfide bond and N-terminal, C
End has marked.
Figure 15 shows recombination MarTX toxin to the depression effect of (alpha+beta 4) BK channel current.
(A) for recombination MarTX toxin to the full cell currents of (alpha+beta 4) BK channel HEK293T, command potential is -80mV, electricity
Stream is induced by the voltage stimulation of+80mV and the interior calcium of 300nM.(B) recombination MarTX toxin inhibit the channel (alpha+beta 4) BK dosage according to
Rely curve, IfIndicate the aftercurrent percentage under each concentration detoxifying function, curve is by Hill equation model.
Specific embodiment
The present inventor after extensive and in-depth study, by largely screening, unexpectedly develops a kind of purifying preparation
The method of MarTX polypeptide.Specifically, in the building of the recombinant plasmid of MarTX, genetic fragment corresponding to target protein
5 ' ends are added coding His label and encode the gene order of MBP label, and the recombinant plasmid transformed that building is obtained is extremely
Carrier bacterial strain E.coli Origami B (DE3), and carry out culture and inducing expression.To the table for the MarTX polypeptide that purifying obtains
Levies in kind test statistics indicate that, the target protein obtained with the method, compared to comparison the resulting target protein of embodiment, have
Higher expression quantity, purity and activity.The present invention is completed on this basis.
Term
Martentoxin albumen and its coded sequence
As used herein, " Martentoxin albumen ", " MarTX toxin " and " MarTX polypeptide " " recombination MarTX toxin "
It is used interchangeably, refers to Martentoxin albumen, by 37 Amino acid profiles, there are three pairs of disulfide bond, be that a species specificity is made
For the short chain polypeptides toxin in the channel BK, nervous system type (alpha+beta 4) BK channel current can be significantly inhibited.
It should be understood that although the gene source provided in example of the invention is derived from other similar in buthus martensii Karscs
Species (the scorpion class for especially belonging to same section or category with buthus martensii Karscs), (preferably, sequence is such as with sequence of the invention
Shown in SEQ ID NO:1) with certain homology (conservative) MarTX gene order, be also included within the scope of the present invention
It is interior, if those skilled in the art after having read the application according to information provided by the present application can be convenient from other species
Isolated sequence in (especially scorpion class).
Polynucleotides of the invention can be DNA form or rna form.DNA form includes: DNA, genomic DNA or people
The DNA of work synthesis, DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide
Coding region sequence can variant identical as coding region sequence shown in SEQ ID NO:1 or degeneracy.
The polynucleotides of encoding mature polypeptide include: the coded sequence of an encoding mature polypeptide；The code sequence of mature polypeptide
Column and various additional coding sequences；The coded sequence (and optional additional coding sequence) and non-coding sequence of mature polypeptide.
The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include
The polynucleotides of additional code and/or non-coding sequence.The invention further relates to the variants of above-mentioned polynucleotides, encode and this
Invention has more glycosides of identical amino acid sequence or segment, the analogs and derivatives of polypeptide.The variant of this polynucleotides can
To be the variant of the allelic variant naturally occurred or non-natural generation.These nucleotide variants include substitution variants,
Deletion variants and insertion variant.As known in the art, allelic variant is the alternative forms of a polynucleotides, it can
It can be substitution, missing or the insertion of one or more nucleotide, but not from the function of substantially changing the polypeptide that it is encoded.
The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least
70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more
The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature
Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) first phthalein when (2) hybridization
Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90% with
On, more preferably 95% or more when, just hybridizes.
It should be understood that although MarTX gene of the invention is preferred from buthus martensii Karscs, (especially from other species
Scorpion class) with buthus martensii Karscs MarTX gene very high homology (as have 80% or more, such as 85%, 90%, 95% or even 98% sequence
The column phase same sex) other genes also within the scope of the present invention contemplates.The Method and kit for of the aligned sequences phase same sex is also this
Field is known, such as BLAST.
MarTX nucleotide full length sequence or its segment of the invention can usually use PCR amplification method, recombination method or artificial conjunction
At method obtain.It, can disclosed related nucleotide sequence, especially open reading according to the present invention for PCR amplification method
Frame sequence carrys out design primer, and with commercially available DNA library or by cDNA prepared by conventional method well known by persons skilled in the art
Library expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly PCR amplification, then again
The segment that each time amplifies is stitched together by proper order.Once obtaining related sequence, so that it may with recombination method come
Related sequence is obtained in large quantity.It is usually cloned into carrier, then is transferred to cell, then by conventional method after proliferation
Host cell in isolated related sequence.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical
After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.At present, it is already possible to passing through completely
Synthesis is learned to obtain encoding the DNA sequence dna of albumen of the present invention (or its segment, or derivatives thereof).Then the DNA sequence dna can be drawn
Enter in various existing DNA moleculars (or such as carrier) as known in the art and cell.In addition, can will also be dashed forward by chemical synthesis
Change is introduced into protein sequence of the present invention.
The present invention relates to a kind of for treating the MarTX polypeptide and its variant of epilepsy, in a preference of the invention,
The amino acid sequence of the polypeptide is as shown in SEQ ID NO:2.Epilepsy can effectively be treated and/or be prevented to polypeptide of the invention.
The invention also includes with sequence shown in SEQ ID NO:2 of the invention have 50% or more (preferably 60% or more,
70% or more, 80% or more, more preferable 90% or more, more preferable 95% or more, most preferably 98% or more, such as 99%) homology
More peptide or proteins with same or similar function.
" same or similar function " is primarily referred to as: " symptom for alleviating epilepsy ".
Polypeptide of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.Polypeptide of the invention can be natural pure
The product of change or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (for example, bacterium, yeast, plant
Object, insect and mammalian cell) in generate.According to host used in recombinant production scheme, polypeptide of the invention can be sugar
Base, or can be nonglycosylated.Polypeptide of the invention may also include or not include the methionine residues of starting.
The invention also includes MarTX polypeptide fragments and analog with MarTX polypeptide active.As used herein, term
" segment " and " analog ", which refers to, is kept substantially natural MarTX polypeptide identical biological function or active more of the invention
Peptide.
Polypeptide fragment of the invention, derivative or the like may is that (i) has one or more conservative or non-conservation ammonia
Base acid residue (preferably conservative amino acid) substituted polypeptide, and can be can also for such substituted amino acid residue
Not to be by genetic code encoding；Or (ii) has the polypeptide of substituent group in one or more amino acid residues；Or
(iii) mature polypeptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol) fusion institute's shape
At polypeptide；Or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide (such as leader sequence or secretion that are formed
Sequence or for purifying the sequence of this polypeptide or proprotein sequence or fusion protein).According to the definition of this paper these segments, spread out
Biology and analog belong to scope known to those skilled in the art.
In the present invention, the polypeptide variants are the amino acid sequences as shown in SEQ ID NO.:2, (logical by several
It is often 1-10, preferably 1-8, more preferably 1-4, most preferably 1-2) replace, miss or add at least one amino acid
Resulting derived sequence, and C-terminal and/or N-terminal addition it is one or several (usually within 10, preferably 5
Within, more preferably it is within 3) amino acid.For example, being taken in the albumen with amino acid similar in performance
Dai Shi does not usually change the function of protein, in one or several (such as 1-3) amino acid of C-terminal and/or N-terminal addition
The function of protein will not generally also be changed.These conservative variations are replaced preferably based on table 1 and are generated.
Table 1
| Initial residue | Representative substitution | It is preferred to replace |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The invention also includes the analogs of albumen claimed.These analogs and natural SEQ ID NO:2 difference
It can be the difference on amino acid sequence, be also possible to not influence the difference on the modified forms of sequence, or have both at the same time.This
The analog of a little albumen includes natural or induction genetic variant.Induction variant can be obtained by various technologies, such as logical
Overshoot is exposed to mutagens and generates random mutagenesis, can also pass through the skill of site-directed mutagenesis or other known molecular biology
Art.Analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and has non-day
So analog of existing or synthesis amino acid (such as β, gamma-amino acid).It should be understood that albumen of the invention be not limited to it is above-mentioned
The representative albumen enumerated.
Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetoxylation of internal or external albumen
Or carboxylated.Modification further includes glycosylation, is carried out such as those in protein synthesis and in processing glycosylation modified.This modification can
To be completed and carrying out glycosylated enzyme (glycosylase or deglycosylation enzyme of such as mammal) by the way that albumen to be exposed to.Modification
Form further includes the sequence with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine).
Fusion protein of the present invention
The present invention provides a kind of fusion proteins, which is characterized in that the fusion protein has shown in Formulas I from N-terminal to C-terminal
Structure:
P1-P2-P3-P4 (Formulas I)
In formula,
P1 is purification tag element；
P2 is maltose-binding protein element (MBP)；
P3 is protease site element；
P4 is MarTX polypeptide element；
"-" indicates the peptide bond of connection said elements.
In one preferred embodiment, the fusion protein may not include the purification tag element of the P1, only
The purifying of Amylose Resin chromatographic column can also be carried out using the maltose-binding protein element of the P2.Include or does not wrap
The difference for including the P1 element is only that the efficiency of purifying difference, but no matter includes or do not include the P1 element, this hair
The conformational stability of expression quantity and gained albumen of the bright fusion protein in host cell of the present invention is significantly better than existing
The other forms of existing recombination MarTX toxin in technology.
In a preferred embodiment, the protease is selected from the group: HRV HRV 3CP, Thrombin enzyme,
Factor Xa, enterokinase (Enterkinase), TEV enzyme, Prescission, TAGZyme, SUMO protease, or combinations thereof.
In another preferred example, the protease is Thrombin enzyme.
In other preferred embodiments, the sequence of the fusion protein further include: P2-P1-P3-P4, P4-P3-P2-
P1, P2-P3-P4-P1 or P4-P3-P1-P2.That is, the sequence between the sequence of His label, MBP label and MarTX polypeptide can
Flexibly change, only need to guarantee restriction enzyme site corresponding to the P3 element close to MarTX polypeptide, and be located at MarTX polypeptide and MBP
Between label, so that in MarTX polypeptide after protease digestion, not comprising MBP label described in P2.And it is final
Resulting MarTX polypeptide may include His label, because His label is only 6 and His residue, existing be will not influence
The activity and structure of MarTX polypeptide.
Preparation method of the present invention
It is provided by the invention to purify the method for preparing MarTX polypeptide, comprising steps of
(i) the fusion protein digestion described to the first aspect of the present invention with protease, so that digestion products are obtained, the digestion
Product corresponds to MarTX polypeptide；With
(ii) isolated or purified goes out the MarTX polypeptide from digestion products.
It in the present invention, is in carrier bacterial strain E.coli Origami B (DE3) for the expression of the fusion protein
It carries out, the condition of culture and inducing expression, is the culture of the bacterial strain and the normal condition for expressing albumen.
In one preferred embodiment, the P1 be His label, be used for by the fusion protein nickel column into
Row affinity chromatography.
In one preferred embodiment, there are the enzymes of one section of Thrombin in the P2 and MarTX polypeptide sequence
Enzyme site carries out staying overnight digestion using the Thrombin enzyme of 6U/mL, and digestion stirs protein solution in 3.5kDa bag filter simultaneously
It mixes overnight.
In one preferred embodiment, the P2 is MBP label, is used to use fusion protein digestion product
Amylose Resin chromatographic column is purified.It in this step, is that the P1-P2 that will be scaled off is incorporated on affinity column,
And target protein MarTX polypeptide is flowed.Percolation ingredient is collected, to obtain the MarTX polypeptide of not tape label.
In one preferred embodiment, further include the steps that gel chromatographic columns are purified, i.e. molecular sieve.At this
In step, collecting retention volume is all eluting peak of 110mL, as the not MarTX polypeptide protein peak of tape label.Through this step
Suddenly after purification, it can get the purer MarTX polypeptide of purity.
Main advantages of the present invention include:
1) other expression plasmids and purification process are compared, the recombination MarTX Output of toxin that the present invention obtains is higher, 2L thallus
About 3mg polypeptide can be obtained, compared to existing other MarTX toxin expression and purification technologies, nearly 10 times of output increased.
2) the method for the present invention prepares resulting MarTX polypeptide, and purity is high, various aspects physicochemical property are correct, while having electricity
Physiological activity.Since natural MarTX Output of toxin is low, recombination MarTX toxin that is difficult, therefore can obtaining with the patent is obtained
Natural MarTX toxin is substituted be of great significance to the research of the channel BK pharmacological mechanism etc..
3) recombinant expression method of the invention overcomes MarTX toxin since polypeptide molecular weight is smaller, and contains multipair disulfide bond
Inclusion body expression is easily led to, so that more difficult obtain a large amount of, soluble, high purity protein defect.
4) the method for the present invention realizes the green manufacturing of MarTX polypeptide, can prepare and provide for the production of similar polypeptide drug
New approaches become the research hotspot of pharmaceutical synthesis and Field of Fine Chemicals.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
Unless otherwise instructed, material and reagent used in embodiment are commercial product.
Embodiment 1:MarTX construction of recombinant plasmid
MarTX (Martentoxin) plasmid is provided by Ji Yonghua seminar, Shanghai University, and totally 37 amino acid, passes through
ProtParam online software predicts that MarTX, relative molecular weight is about 4.065kDa, Theoretical pi 8.65.Gene piece
Section is located between BamH I and Sma the I cloning site of pGEX-4T-3 carrier, the GST label of amalgamation and expression and desired polypeptides it
Between there are enterokinase cleavage site (Fig. 5).
Host strain E.coli DH5 α, BL21 (DE3) and Origami B (DE3) strain are purchased from Novagen company, rear equal
It is independently prepared by this laboratory.
The building of 1.1pGEX-6P-1-MarTX recombinant plasmid
A) PCR reacts
PCR primer is designed first, adds BamH I and Xho I restriction enzyme site at 5 ' ends of MarTX genetic fragment and 3 ' ends
With protection base, PCR reaction is carried out by template of pGEX-4T-3-MarTX recombinant plasmid, it is solidifying by 1% agarose after the completion
Gel electrophoresis identification.It is single that electrophoretogram shows that purpose band becomes clear, PCR success (Fig. 2).Then carry out the rubber tapping of target gene fragment
Recovery operation.
B) pGEX-6P-1 carrier and the double digestion of genetic fragment, connection reaction
25 μ L PCR recovery products and pGEX-6P-1 carrier solution are taken respectively, and 3 μ L10 × K of Takara company are added
Buffer, BamH I and Xho I restriction endonuclease 30 μ L double digestion systems of each 1 μ L composition, the digestion 2 hours in 37 DEG C of water-baths, it
After carry out 1% agarose gel electrophoresis identification and be tapped and recovered.
Carrier 0.5 μ L, MarTX after double digestion is added in 200 μ L sterilizing EP pipe cut 4.5 μ L of post-fragment, and T4 is added and connects
Meet enzyme 1 μ L, ddH23 μ L of O forms 10 μ L linked systems, is placed in 16 DEG C of metal baths and connects overnight.
C) it converts, identification is sequenced
After the completion of connection, it is transferred in amplification bacterial strain E.coli DH5 α competence, step of converting is as follows:
1) the 100 μ L of Efficiency Competent Cells DH5 α for taking out -80 DEG C of preservations, is placed in slow defrosting 10min on ice；
2) 5-10 μ L connection reaction solution is taken to add in competent cell, gently piping and druming mixes, and is placed in 30min on ice；
3) 42 DEG C of water-bath heat shock 90s, are immediately placed in 2-3min on ice；
4) 800 μ L LB culture solutions are added into pipe, in 37 DEG C of 190rpm shaken cultivation 40min；
5) 3000rpm is centrifuged 10min, discards most of culture solution, so that cell is suspended in remaining culture solution, be seeded to
On solid LB media containing corresponding antibiotic, coating is uniform；
6) the solid culture ware is stayed overnight in 37 DEG C of culture carton upside downs, generally culture 14-16h.
The uniform bacterium colony of next day picking is cultivated, served after bacterium solution is muddy Hai Boshang Bioisystech Co., Ltd into
Row sequencing identification.By above step, we successfully construct pGEX-6P-1-MarTX recombinant plasmid.
The building of 1.2 pSMT3-MarTX recombinant plasmids
For pSMT3-MarTX recombinant plasmid, by between genetic fragment insertion BamH I and the Xho I of toxin, this with
The cloning site of pGEX-6P-1-MarTX recombinant plasmid is identical.Therefore, pass through identical operating procedure: PCR reaction, pSMT3 are carried
Body and the double digestion (BamH I and Xho I restriction endonuclease) of genetic fragment, connection reaction and conversion, sequencing are identified and are successfully constructed
PSMT3-MarTX recombinant plasmid.
The building of 1.3 pETDuet-1-MarTX recombinant plasmids
There are two multiple cloning sites MCS1 and MCS2 in pETDuet-1 plasmid.By the protein-bonded MBP of encoding mannose
Label is implemented between Nco I and the EcoR I site of MCS1, while designing 6 His-tag in the N-terminal of MBP label.It is logical first
It crosses PCR reaction and Thrombin restriction enzyme site and EcoR I cloning site is added in 5 ' ends of MarTX genetic fragment, be added in 3 ' ends
Not I cloning site, design of primers are as shown in table 2.
The design of primers of 2 pETDuet-1-MarTX recombinant plasmid of table
The step of according to 1.1, completes PCR reaction by template of pGEX-4T-3-MarTX recombinant plasmid, then carries out
PETDuet-1 plasmid and the double digestion (EcoR I and Not I restriction endonuclease) of MarTX genetic fragment, connection reaction and conversion are surveyed
Sequence identification, successfully constructs pETDuet-1-MarTX recombinant plasmid.
Embodiment 2: the prokaryotic expression of recombination MarTX toxin
For pETDuet-1-MarTX recombinant plasmid, be transferred in E.coli Origami B (DE3) expression bacterial strain into
Row expression.The present invention selects E.coli Origami B (DE3) as expression bacterial strain.
Steps are as follows:
E.coli Origami B (DE3) competent cell is prepared, it will sequencing using heat shock (42 DEG C heat shock 90 seconds)
Correct recombinant expression carrier pETDuet-1-MarTX is converted into Origami B (DE3), obtains the table containing recombinant plasmid
Up to bacterial strain.The step of efficient prokaryotic expression, is as follows:
1) test tube that expression bacterial strain monoclonal is seeded to the culture medium of LB containing 5mL (antibiotic of benzyl containing ammonia) is picked from the plate
In, 37 DEG C, 220rpm overnight incubation；
2) next day shakes 5mL in muddy bacterium solution access 100mL (antibiotic of benzyl containing ammonia) in bottle LB culture solution, 37 DEG C,
220rpm shaken cultivation 3h；
3) take wherein the 20mL 37 DEG C of 220rpm in 1L (antibiotic of benzyl containing ammonia) LB culture solution that transfer cultivate to OD600It reaches
It is cooling with ice-water bath when 0.6-0.8 or so, IPTG to final concentration of 0.5mM is added, in 18 DEG C of 220rpm inducing expressions；
4) after expression 20h, 18 DEG C, 5000rpm centrifugation 15min collection thallus abandoning supernatant, -80 DEG C are saved for use.
Similarly, pGEX-4T-3-MarTX recombinant plasmid is transferred in E.coli BL21 (DE3) host cell and carries out table
It reaches；For pGEX-6P-1-MarTX recombinant plasmid, it is transferred in E.coli BL21 (DE3) expression bacterial strain and is expressed；It is right
In pSMT3-MarTX recombinant plasmid, which is transferred in E.coli BL21 (DE3) competent cell and is expressed.
Embodiment 3: the purifying preparation of recombination MarTX toxin
3.1 purification step
For the fusion protein of pETDuet-1-MarTX expression of recombinant plasmid, i.e. MBP label recombinates MarTX polypeptide, and every liter
Thallus is added 15mL and breaks bacterium Buffer A (see Table 2 for details for ingredient), and protease inhibitors PMSF is added after being resuspended uniformly to final concentration
For 0.1mM.Bacterium instrument is crushed with height and is cooled to 4 DEG C or so broken bacterium, and 1000bar or so is arranged in pressure, repeats broken bacterium 3 times.By broken bacterium
Liquid is collected into centrifuge tube, after 4 DEG C of 16000rpm centrifugation 50min, collects the supernatant containing recombination fusion protein.
Steps are as follows for specific purification process:
1) fusion protein in the Buffer A environment is first with nickel column in conjunction with, then use Buffer B imidazoles salting liquid into
Row gradient elution removes most of foreign protein, completes first affine column purification and carries out SDS-PAGE electrophoresis detection, referring to attached
Fig. 8 A；
2) it collects purpose fusion protein (about 46.8kDa) and carries out dialysis digestion in 18 DEG C, 2L Buffer C, according to 6U/
Thrombin enzyme is added in mL, and digestion is stirred in 3.5kDa bag filter and is stayed overnight；
3) then digestion mixture is loaded on Amylose Resin chromatographic column with constant flow pump, collects percolation ingredient
(FL) and Buffer D elutes ingredient.His-MBP-tag is eluted through Buffer E and is removed, and is completed second affine column purification and is gone forward side by side
Row SDS-PAGE electrophoresis detection, referring to attached drawing 8B；
4) then by FL ingredient and Buffer D cleaning solution (usually containing a small amount of recombination MarTX toxin) be concentrated into 2mL into
Row next step gel chromatography column purification；
5) gel chromatographic columns Superdex 75 is balanced with Buffer F in advance, and then sample is loaded on pillar, is used
Buffer F is eluted.Collecting the eluting peak that retention volume is 110mL or so is to recombinate MarTX toxin sample, before
Absorption peak is MBP-tag or the fusion protein not cut.UV absorption peak figure is referring to attached drawing 8C；
6) SDS-PAGE detects purification effect, referring to attached drawing 8D.Relevant buffers component such as 3 institute of table in purification process
Show.
Relevant buffers during the recombination of table 3 MarTX is toxin Purified
| Buffer title | Buffer composition |
| Buffer A | 25mM Tris,500mM NaCl,pH 7.50 |
| Buffer B | 25mM Tris,500mM NaCl,500mM imidazole,pH 7.50 |
| Buffer C | 25mM Tris,150mM NaCl,pH 7.50 |
| Buffer D | 25mM Tris,150mM NaCl and 0.5mM EDTA,pH 7.50 |
| Buffer E | 25mM Tris,150mM NaCl and 0.5mM EDTA,300mM Maltose,pH 7.50 |
| Buffer F | 25mM NaH2PO4,100mM NaCl,pH 6.80 |
To recombinant protein expressed by the other recombinant plasmids constructed in embodiment 1, using similar corresponding way of purification.
For the fusion protein of pGEX-4T-3-MarTX expression of recombinant plasmid, first will when having GST label, therefore purifying
Then albumen carries out gradient elution with reduced glutathione solution and removes most of foreign protein in conjunction with GST column.Later in room
Temperature is lower to carry out enterokinase digestion removal GST label, then carries out second of GST column and a gel chromatography column purification, to obtain
MarTX polypeptide.
For the fusion protein of pGEX-6P-1-MarTX expression of recombinant plasmid, a GST label is had, first by albumen when purifying
In conjunction with GST column, gradient elution then is carried out with reduced glutathione solution and removes most of foreign protein.Later at 4 DEG C
It carries out the digestion of 3C enzyme to separate polypeptide and GST label, then carries out second of GST column and a gel chromatography column purification, thus
To MarTX polypeptide.
For the fusion protein of pSMT3-MarTX expression of recombinant plasmid, first by albumen when having His label, therefore purifying
In conjunction with nickel column, gradient elution then is carried out with imidazoles salting liquid and removes most of foreign protein.UlpI enzyme is carried out at 4 DEG C later
Digestion, then second of ni-sepharose purification and a gel chromatography column purification are carried out, to obtain MarTX polypeptide.
3.2 purification result
For the fusion protein of pGEX-4T-3-MarTX expression of recombinant plasmid, by touching for different inducing temperatures and time
Rope (20h or 28 DEG C of induction 5h of 18 DEG C of inductions), and the enterokinase of different manufacturers has been attempted, all fail to obtain sufficient amount
MarTX carries out subsequent experimental study, and 2L thallus at most can only obtain about 0.3mg polypeptide, and expression quantity is too low (Fig. 5).Therefore it needs
Recombinant plasmid is advanced optimized, its genetic fragment is implemented in other carriers by trial, and is changed to expression and purification method
Into.
For the fusion protein of pGEX-6P-1-MarTX expression of recombinant plasmid, it has been found that 3C enzyme is not easy fusion protein
GST label and toxin cut, the MarTX obtained after digestion is still less, the failure of an experiment (Fig. 6).
For the fusion protein of pSMT3-MarTX expression of recombinant plasmid, under the conditions of first time ni-sepharose purification, 4 DEG C
The digestion of UlpI enzyme, second of ni-sepharose purification and gel chromatography column purification, the recombinant plasmid also fail to obtain purpose toxin, and experiment is again
Primary failure (Fig. 7).
However, the fusion protein of pETDuet-1-MarTX expression of recombinant plasmid is expressed above and after purification, is obtained
The higher recombination MarTX toxin of yield and purity was obtained, the thallus of 2L LB culture medium can obtain about 3mg polypeptide, referring to attached drawing
8C.Compared to the pGEX-4T-3-MarTX expression and purification scheme that Shanghai University provides, 2L thallus can obtain about 3mg polypeptide, and yield mentions
It is nearly 10 times high, meet the sample requirement of subsequent experimental toxin.
Embodiment 4: the characterization of recombination MarTX toxin
In order to which whether the recombination MarTX toxin further confirmed that is correct, to the molecular weight and second level of recombinant toxin
Structure is verified, while having investigated the folded situation of toxin in the solution using one-dimensional hydrogen spectrum.
4.1 recombination MarTX toxin1H NMR
Recombination MarTX concentration is 0.42mM, in 25mM NaH2It is added in the Buffer of PO4,100mM NaCl, pH 6.80
10% D2O acquires one-dimensional hydrogen spectrum on 600MHz nmr spectrometer, and sample temperature is 20 DEG C, tests spectrogram referring to attached drawing 9.
The nuclear magnetic signal of the recombination MarTX toxin of acquisition compares diverging, there is apparent methyl characteristic peak near high field region,
And the acylamino hydrogen and aromatic ring hydrogen of toxin are widely distributed in the low field area of 6.5-10.5ppm, illustrate that polypeptide is not assembled, space knot
Structure folds good.
The circular dichroism spectra of 4.2 recombination MarTX toxin
In order to further verify to the secondary structure of toxin, the circular dichroism spectra (CD spectrum) of recombination MarTX is determined.
The Buffer condition of toxin is exchanged to ddH2In O, sample concentration 0.5mg/mL, experimental result is referring to attached drawing 10.
It follows that recombinant toxin of the invention includes the α spiral and β-pleated sheet Secondary structural elements for having delivered structure, say
It is bright to fold normally.
The quality determination of 4.3 recombination MarTX toxin
Toxin sample Buffer condition after purification is exchanged to ddH2It in O, is measured through MALDI-TOF, recombinates MarTX's
Molecular weight is 4200.5444, referring to attached drawing 11.Recombinate more two amino acid of GS of MarTX its N-terminal after Thrombin digestion, reason
It is 4208.80 by molecular weight, therefore the molecular weight and theoretical value that measure are roughly the same.
The solution structure parsing of 4.4 recombination MarTX toxin
The acquisition of two-dimentional NMR spectrogram has been carried out to recombination MarTX sample.It tests and is carried out on Aligent 600MHz spectrometer,
Sample concentration is 1mM, and sample temperature is 20 DEG C, and sampling Buffer is 25mM NaH2PO4, 100mM NaCl, 10%D2O, pH
6.80.The two-dimentional same core NMR spectra of acquisition is as shown in table 4.
Table 4 recombinates the acquisition of MarTX toxin spectrogram and parameter setting
| Experiment | Number of increments | SW(Hz) | Carrier frequency(ppm) |
| DQF_COSY | 1000(H)×256(H) | 6906(H)×6906(H) | 4.82(H)×4.82(H) |
| TOCSY | 1024(H)×256(H) | 9579(H)×6010(H) | 4.82(H)×4.82(H) |
| NOESY | 1024(H)×256(H) | 12019(H)×9597(H) | 4.82(H)×4.82(H) |
Signals assignment is carried out to the above spectrogram, then Structure Calculation is carried out and refine obtains 100 solution structures.From 100
Representative conformation of the structure of 20 minimum energies as recombination MarTX toxin is selected in a refined structure according to energy ordering,
20 optimal solution structures are overlapped, the results showed that other than loop more flexible in toxin, remaining secondary structure portion
Split-phase is to convergence, referring to attached drawing 12.
It is assessed with 20 optimal comformation in solutions of the PROCHECK_NMR software to recombination MarTX toxin, referring to attached
Figure 13.Ramachandran map analysis as the result is shown itsAll in acceptable area, 20 Optimum configurations averagely have for angle and the angle ψ
91.8% residue is in most permission region, and 4.4% residue is in time permission region, and being in without residue does not allow region,
Show structurally reasonable.
Obtained structure and reported natural MarTX (PDB:1M2S) solution structure are overlapped, RMSD value isReferring to attached drawing 14.Show the recombination MarTX toxin structure of nuclear-magnetism parsing and the day extracted from buthus martensii Karscs venom
Right toxin structure is consistent, illustrates that MarTX toxin has successfully been prepared in prokaryotic expression system through the invention, can be used for next
Step is in the investigation of neurotoxin active.
Embodiment 5: inhibition of the recombination MarTX toxin to (alpha+beta 4) BK channel current
Document [Biophys.J.2008,94,3706-3713] shows the born of the same parents of MarTX toxin Yu 4 auxiliary subunit of BK channel β
The outer area loop interaction, to inhibit (alpha+beta 4) BK channel current.The present invention examines recombination by patch clamp experiments
Electrophysiologic activity of the MarTX toxin to the channel (alpha+beta 4) BK.
Experimental procedure: Human embryonic kidney cells (HEK293T) containing 10% heat-inactivated FBS DMEM (Gibco company,
The U.S.) it cultivates in culture solution, growth conditions of the cell in culture dish is 5%CO2, 37 DEG C of constant temperature, constant humidity 95%.Using instantaneous
Rotaring dyeing technology, by lipofection by the channel source of people (alpha+beta 4) BK plasmid (α containing hSlo (U23767), 4 (KCNMB4 of β；
AF207992 gene order)) it is transferred in HEK293 cell and expresses.
Full cell voltage is carried out using EPC-9 amplifier (HEKA Eletronik, Germany) under room temperature (21 DEG C -25 DEG C)
Patch clamp experiments.Extracellular fluid is NaCl 135mM, KCl 5mM, MgCl in experiment21.2mM, CdCl22.5mM, HEPES
5mM, glucose 10mM (adjust pH to 7.4 with NaOH)；Liquid is NaCl 10mM, KCl 117mM, MgSO in electrode42mM,
HEPES 10mM, MgATP 2mM, EGTA 1mM (adjust pH to 7.2 with KOH).
Data processing: data pass through PulseFit 8.5 (HEKA Eletronik, Germany) and Origin8.5
(Northampton company, the U.S.) is analyzed.Experimental result mean+/-standard error (Mean ± S.E.M) expression, n
For experimental cell number.Fraction of current is the steady-state current that drug (the recombination MarTX toxin) channel BK afterwards is added
The ratio of electric current is compareed with when drug not being added.
Experimental result: measuring through the above patch clamp experiments and find, the pronuclear recombination expression system developed using the present invention is obtained
The recombination MarTX toxin obtained has electrophysiologic activity, referring to attached drawing 15A.10 μM of recombination MarTX toxin can significantly inhibit (alpha+beta 4)
BK channel current, If(Fraction of current)=0.33 ± 0.06, n=6, with pharmacological property phase reported in the literature
Symbol.
Embodiment 6: the measurement of the recombination channel MarTX toxin blocks (alpha+beta 4) BK half effective concentration
The electric current generated by the channel (alpha+beta 4) BK can be effectively suppressed in 10 μM of recombination MarTX toxin, and has dose dependent
Feature, referring to attached drawing 15B.The half effective concentration in the channel recombination MarTX toxin blocks (alpha+beta 4) BK that amount effect curve is shown
(EC50) it is 0.32 ± 0.012 μM.
Thus, the recombination MarTX toxin obtained through Prokaryotic expression, purification of the present invention can be realized to the channel (alpha+beta 4) BK
Effective blocking of electric current, can be used as the inhibitor in the channel nervous system type BK, for epilepsy relevant to nervous system type BK channel function etc.
The research of disease.
It discusses
In an embodiment of the present invention, there is the comparative example of three groups of carry out parallel laboratory tests, specifically, respectively by MarTX gene
Being integrated into vector plasmid pGEX-4T-3 (it expresses GST label), pGEX-6P-1 (it expresses GST label) and pSMT3, (it is expressed
His label and SUMO label) in, and expressed in E.coli BL21 (DE3) respectively.
According to the result (referring to embodiment 3) of its expression and purifying respectively, three groups of comparative examples are merged compared to the present invention
The purification result of albumen, obtained protein yield is low or purity is low.
Its reason is analyzed, for pGEX-4T-3-MarTX and pGEX-6P-1-MarTX, contains GST label, GST label
Molecular weight about 26kDa, and purpose toxin only has about 4kDa, and due to the structure feature of GST label, it may be by protease digestion
Site and polypeptide package wherein, make protease cannot achieve digestion function, few so as to cause the desired polypeptides amount after digestion.It is right
In pSMT3-MarTX, expression quantity just seldom, i.e., has just failed in expression step.
The poor reason of induction and contrast example effect, the present inventor, which speculates, may be because that MarTX lps molecule amount is smaller, be not easy
It is cut with fusion tag.And contain three pairs of disulfide bond in toxin, it is correct to be not easy reconstruct.And disulfide bond protein expression system is conducive to
The correct folding of disulfide bond.Origami B (DE3) belongs to escherichia coli cloning type strain, contains trxB/gor gene mutation.Together
When be mutated the two genes the bacterial strain enabled more efficiently to generate disulfide bond in cytoplasm, facilitate containing disulfide bond
Activated protein is formed.
Therefore, it have passed through and largely attempt and grope, the present inventor finally has developed MarTX polypeptide preparation of the invention
Method using recombinant plasmid of the present invention, and uses E.coli Origami B (DE3) bacterial strain, successfully obtains production
Amount and the higher MarTX toxin sample of purity, overcome MarTX toxin since polypeptide molecular weight is smaller, and contain multipair two sulphur
Key easily leads to inclusion body expression, so that more difficult obtain a large amount of, soluble, high purity protein defect.Technical side of the invention
The expression and purification scheme of case existing MarTX toxin compared to the prior art, 2L thallus can obtain about 3mg polypeptide, output increased
Nearly 10 times, realize the efficient green manufacture of MarTX polypeptide.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Shanghai Organic Chemistry Institute, Chinese Academy of Sciences
<120>preparation, characterization and the application of Martentoxin are recombinated
<130> P2018-1737
<160> 6
<170> PatentIn version 3.5
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<212> DNA
<213>buthus martensii Karscs (Buthus martensi Karsch)
<400> 1
tttggactca tagacgtaaa atgttttgca tctagtgaat gttggacagc ttgcaaaaaa 60
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<210> 2
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<212> PRT
<213>buthus martensii Karscs (Buthus martensi Karsch)
<400> 2
Phe Gly Leu Ile Asp Val Lys Cys Phe Ala Ser Ser Glu Cys Trp Thr
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Ala Cys Lys Lys Val Thr Gly Ser Gly Gln Gly Lys Cys Gln Asn Asn
20 25 30
Gln Cys Arg Cys Tyr
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<213>Escherichia coli (Escherichia coli)
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atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60
ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120
ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180
atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240
accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300
aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360
gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420
aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480
ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540
gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600
aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660
ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720
gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780
ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840
ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900
ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960
accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020
tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080
gccctgaaag acgcgcagac t 1101
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<213>Escherichia coli (Escherichia coli)
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Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
<210> 5
<211> 55
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 5
ccggaattcc tggtccctag aggttctttt ggactcatag acgtaaaatg ttttg 55
<210> 6
<211> 45
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 6
ctgcggccgc tcagtagcat cgacattgat tattctggca ctttc 45
Claims (10)
1. a kind of fusion protein, which is characterized in that the fusion protein has structure shown in Formulas I from N-terminal to C-terminal:
P1-P2-P3-P4 (Formulas I)
In formula,
P1 is purification tag element；
P2 is maltose-binding protein element (MBP)；
P3 is protease site element；
P4 is MarTX polypeptide element；
"-" indicates the peptide bond of connection said elements.
2. fusion protein as described in claim 1, which is characterized in that the sequence of the P2 is SEQ ID NO:4.
3. fusion protein as described in claim 1, which is characterized in that the sequence of the P4 is SEQ ID NO:2.
4. a kind of isolated polynucleotides, which is characterized in that described in any one of described polynucleotide encoding claim 1 to 3
Fusion protein.
5. a kind of carrier, which is characterized in that the carrier contains polynucleotides as claimed in claim 4.
6. a kind of host cell, which is characterized in that the host cell contains whole in carrier described in claim 5 or genome
Conjunction have the right to require 4 described in polynucleotides.
7. host cell as claimed in claim 6, which is characterized in that the host cell is E.coli Origami B
(DE3)。
8. a kind of method for generating fusion protein described in any one of claims 1 to 3, which is characterized in that comprising steps of
Under conditions suitable for the expression, host cell as claimed in claim 6 is cultivated, to give expression to described in claim 1
Fusion protein.
9. a kind of method for preparing MarTX polypeptide, which is characterized in that comprising steps of
(i) with protease to fusion protein digestion described in claim 1, to obtain digestion products, the digestion products are corresponding
In MarTX polypeptide；With
(ii) isolated or purified goes out the MarTX polypeptide from digestion products.
10. the drug screening method of one kind (alpha+beta 4) BK channel current inhibitor, which is characterized in that comprising steps of
(i) method as claimed in claim 9, purifying preparation MarTX polypeptide are used；
(ii) using MarTX polypeptide as positive control, (alpha+beta 4) BK channel current inhibitor drug candidate is screened.
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|---|---|---|---|
| CN201811308401.8A CN109384852B (en) | 2018-11-05 | 2018-11-05 | Preparation, characterization and application of recombinant Martentoxin |
| PCT/CN2019/115709 WO2020094005A1 (en) | 2018-11-05 | 2019-11-05 | Preparation, characterization and use of recombinant martentoxin |
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| CN201811308401.8A CN109384852B (en) | 2018-11-05 | 2018-11-05 | Preparation, characterization and application of recombinant Martentoxin |
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| CN109384852B CN109384852B (en) | 2021-09-03 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110590921A (en) * | 2019-08-15 | 2019-12-20 | 武汉大学 | Human Kv1.3 type potassium ion channel activity inhibition peptide mimic, and preparation method and application thereof |
| WO2020094005A1 (en) * | 2018-11-05 | 2020-05-14 | 中国科学院上海有机化学研究所 | Preparation, characterization and use of recombinant martentoxin |
| CN112210569A (en) * | 2020-10-16 | 2021-01-12 | 江苏省中医药研究院 | Recombinant Buthus martensii Karsch polypeptide Makatoxin-3 and preparation method and application of mutant thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1344745A (en) * | 2001-10-12 | 2002-04-17 | 吉永华 | Martentoxin as one great-conductance calcium-activating potassium channel blocker and its prepn and use |
| CN102242125A (en) * | 2011-04-28 | 2011-11-16 | 上海大学 | BK channel blocker gene, recombinant vector thereof and cloning method thereof |
| CN104946677A (en) * | 2015-04-07 | 2015-09-30 | 上海大学 | Recombinant plasmid of specific ligand of BK channel and recombinant expression method thereof |
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| CN109384852B (en) * | 2018-11-05 | 2021-09-03 | 中国科学院上海有机化学研究所 | Preparation, characterization and application of recombinant Martentoxin |
-
2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1344745A (en) * | 2001-10-12 | 2002-04-17 | 吉永华 | Martentoxin as one great-conductance calcium-activating potassium channel blocker and its prepn and use |
| CN102242125A (en) * | 2011-04-28 | 2011-11-16 | 上海大学 | BK channel blocker gene, recombinant vector thereof and cloning method thereof |
| CN104946677A (en) * | 2015-04-07 | 2015-09-30 | 上海大学 | Recombinant plasmid of specific ligand of BK channel and recombinant expression method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈孝明: "Martentoxin的表达体系优化及其与BK通道孔区的交互结构模拟", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020094005A1 (en) * | 2018-11-05 | 2020-05-14 | 中国科学院上海有机化学研究所 | Preparation, characterization and use of recombinant martentoxin |
| CN110590921A (en) * | 2019-08-15 | 2019-12-20 | 武汉大学 | Human Kv1.3 type potassium ion channel activity inhibition peptide mimic, and preparation method and application thereof |
| CN112210569A (en) * | 2020-10-16 | 2021-01-12 | 江苏省中医药研究院 | Recombinant Buthus martensii Karsch polypeptide Makatoxin-3 and preparation method and application of mutant thereof |
| CN112210569B (en) * | 2020-10-16 | 2022-08-26 | 江苏省中医药研究院 | Recombinant Buthus martensii Katoxin polypeptide Makatoxin-3 and preparation method and application of mutant thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109384852B (en) | 2021-09-03 |
| WO2020094005A1 (en) | 2020-05-14 |
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