CN110078791A - A method of it is identified based on amino acid specificities and realizes protein cross - Google Patents
A method of it is identified based on amino acid specificities and realizes protein cross Download PDFInfo
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- CN110078791A CN110078791A CN201910405266.7A CN201910405266A CN110078791A CN 110078791 A CN110078791 A CN 110078791A CN 201910405266 A CN201910405266 A CN 201910405266A CN 110078791 A CN110078791 A CN 110078791A
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Abstract
The present invention relates to a kind of peptide chain label for realizing protein cross, peptide chain length is 8 to 15 amino acid, and at least provided with a lysine and/or glutamine in peptide chain, amino acid sequence includes at least one of Sequence No.1-4.The invention further relates to a kind of method for realizing protein cross, the N-terminal of the peptide chain gene insertion protein with the two designated amino acids of lysine or glutamine is passed through the corresponding protein of Bacillus coli expression by this method.The function of thering is specific recognition to be crosslinked the primary amine group of lysine and the amide group of glutamine using glutamine transaminage (TGase), it realizes the orderly crosslinking between protein while can guarantee the active without significantly sacrificing of protein, be with a wide range of applications.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of side for identifying based on amino acid specificities and realizing protein cross
Method realizes protein cross more particularly to a kind of peptide chain label for realizing protein cross and based on the peptide chain label
Method.
Background technique
Glutamine transaminage (TGase) is found in the liver of cavy by Clarke et al. earliest, is then existed again
Animals and plants, fish find in microorganism.Glutamine transaminage (TGase) can catalytic proteins or polypeptide glutamine it is residual
Acyl group transfer reaction occurs between base and primary amine group.As microorganism produces the maturation of glutamine transaminage (TGase) technology
With commercially produce, glutamine transaminage (TGase) is applied in more and more fields, as field of food (meat,
The food processings such as milk, beans), biomedicine field (pharmaceutical carrier, medical stent), Material Field etc..Glutamine turns ammonia
Enzyme (TGase) causes many concerns in terms of the functionality of modification protein and enzyme recently.Noriho Kamiya by using
Glutamine transaminage cross-linked lipase crude enzyme liquid, it was demonstrated that other than rizolipase crude enzyme liquid, remaining lipase can not be crosslinked,
And it will find that pure enzyme can not be crosslinked after head mold fat ni-sepharose purification.
Therefore, presently, there are the problem of be need to research and develop it is a kind of using glutamine transaminage realize protein it is orderly
Crosslinking while the active method without significantly sacrificing that can guarantee protein.
Summary of the invention
It is a kind of with specific amino acids the technical problem to be solved by the present invention is in view of the deficiencies of the prior art, provide
Peptide chain label, the deoxyribonucleotide sequence insertion protein gene of the peptide chain label is obtained with the peptide after expressing
The albumen of chain label can realize protein cross using glutamine transaminage.Glutamy is utilized the present invention also provides a kind of
The gene of the above-mentioned peptide chain label with specific amino acids is inserted into egg by the method that amine transaminase realizes protein cross, this method
White gene carries out expression culture, and the albumen obtained with the peptide chain label realizes that protein has using glutamine transaminage
Sequence crosslinking, while can guarantee the activity of protein without significantly sacrificing.
For this purpose, first aspect present invention provides a kind of peptide chain label for realizing protein cross, peptide chain length
For 8 to 15 amino acid, include at least provided with a lysine and/or glutamine, amino acid sequence in peptide chain
At least one of Sequence No.1-4.
According to the present invention, in the peptide chain, the site of lysine and/or glutamine is close to N-terminal.
In some embodiments of the invention, the deoxyribonucleotide sequence of the peptide chain includes Sequence No.5-
At least one of 8.
Second aspect of the present invention provides a kind of method for realizing protein cross comprising:
The deoxyribonucleotide sequence insertion protein gene segment 5 ' of peptide chain label is held, obtains and have peptide by step L
The protein gene segment of the deoxyribonucleotide sequence of chain label；
Step M, the protein gene segment of deoxyribonucleotide sequence of the expression with peptide chain label, obtains and has peptide
The protein of chain label；
Step N is crosslinked the protein with peptide chain label under glutamine transaminage (TGase) existence condition
Crosslinking protein is made；
Wherein, the peptide chain label is the peptide chain label as described in any one of claim 1-3.
According to the present invention, in step N, glutamine transaminage is added to the bottom containing the protein with peptide chain label
In object solution, cross-linking reaction is carried out after mixing, and crosslinking protein is made.
In some embodiments of the invention, the usage amount of glutamine transaminage is in terms of the volume of substrate solution
1wt%-3wt%.
In the present invention, the substrate solution is formed by being dissolved in buffer solution containing the protein with peptide chain label.
In some embodiments of the invention, the content of the protein with substrate solution total volume meter with peptide chain label is
5-10mg/mL。
In some preferred embodiments of the invention, the buffer solution is the Tris-HCl solution of 0.05mol/L.
In some embodiments of the invention, the temperature of the crosslinking is 20-30 DEG C.
In some embodiments of the invention, crosslinking time is 4-12 hours.
According to the present invention, the step M includes:
The genetic fragment of the protein of deoxyribonucleotide sequence with peptide chain label is connected to carrier by step B
Upper building recombinant expression carrier；
Step C, recombinant expression carrier are transferred to host cell, obtain expression bacterial strain；
Expression bacterial strain is carried out fermented and cultured, and carries out inducing expression with inducer by step D, after thallus is then broken born of the same parents
Again it suspends, is centrifuged, collect supernatant and isolated and purified, obtain the protein sterling for having peptide chain label.
In the present invention, the host cell is Escherichia coli, and the preferably described host cell is e. coli bl21 (DE3).
In the present invention, the carrier is pET32b (+) carrier.
In the present invention, the inducer is IPTG.
In some embodiments of the invention, the additional amount of the inducer is the 0.05wt% of fermentating liquid volume.
According to the present invention, in step D, fermented and cultured will be carried out in expression bacterial strain access culture medium, induction is then added
Agent carries out inducing expression culture.
Preferred embodiment in accordance with the present invention will carry out fermented and cultured in expression bacterial strain access culture medium, work as OD600
When reaching 0.8, inducer is added and carries out inducing expression culture.
In the present invention, the culture medium is LB culture medium or TB culture medium.
In some embodiments of the invention, the temperature of fermented and cultured is 37 DEG C.
In some embodiments of the invention, the temperature of inducing expression culture is 15-25 DEG C.
In some specific preferred embodiments of the invention, fermented and cultured is carried out in shaking table.
It is 180rpm in the shaking speed of the invention in some specific preferred embodiments of the invention.
In some specific preferred embodiments of the invention, inducing expression culture is carried out in shaking table.
In some specific preferred embodiments of the invention, the shaking speed is 180rpm.
In some specific preferred embodiments of the invention, the time of the inducing expression culture is 12-20 hours.
According to the present invention, in step D, the method isolated and purified includes affinity purification.
In some preferred embodiments of the invention, affinity purification is carried out using nickel column.
In some specific preferred embodiments of the invention, during affinity purification, using the Tris- of 0.05mol/L
HCl solution removes foreign protein, is eluted using 0.1mol/L imidazole solution.
The present invention is that the protein of peptide chain label is had by Bacillus coli expression using technique for gene engineering, is recycled
Glutamine transaminage (TGase) realizes protein cross to the specific recognition of amino acid in peptide chain.This method have with
Lower advantage:
(1) the peptide chain modification that protein is realized in gene level, does not need to modify protein in vitro again；
(2) biocatalyst is used --- glutamine transaminage (TGase) crosslinking has the protein of peptide chain label, tool
There is the function of specific recognition；
(3) glutamine transaminage (TGase) will not become a part of cross-linking products, and glutaraldehyde, Geniposide etc. are changed
Can then a part of product be become by learning crosslinking agent, therefore the recycling of glutamine transaminage (TGase) may be implemented；
(4) protein molecular is transformed in gene level, so that its included peptide chain, peptide chain is crosslinked, will not influence protein
The structure and performance of molecule itself.
Detailed description of the invention
It is next with reference to the accompanying drawing that invention is further described in detail:
The crosslinking of Fig. 1 glutamine transaminage has the enhanced green fluorescence protein of S peptide tag.
The crosslinking of Fig. 2 glutamine transaminage has the enhanced green fluorescence protein of F peptide tag and M peptide tag.
Fig. 3 glutamine transaminage is crosslinked the albumen with the carbonic anhydrase of F peptide tag and the hydrogenlyase of M peptide tag
Electrophoretogram.
Fig. 4 glutamine transaminage is crosslinked the enzyme activity with the carbonic anhydrase of F peptide tag and the hydrogenlyase of M peptide tag
As a result.
Fig. 5 glutamine transaminage is crosslinked the egg with the carbonic anhydrase of K2 peptide tag and the hydrogenlyase of M peptide tag
White electrophoretogram.
Fig. 6 glutamine transaminage is crosslinked the enzyme with the carbonic anhydrase of K2 peptide tag and the hydrogenlyase of M peptide tag
Slip-knot fruit.
Specific embodiment
To be readily appreciated that the present invention, below in conjunction with attached drawing, the present invention will be described in detail.But before describing the present invention in detail,
It should be understood that the present invention is not limited to the specific embodiments of description.It is also understood that term used herein is only for description
Specific embodiment, and be not offered as restrictive.
Unless otherwise defined, all terms used herein and those skilled in the art's is usual
Understand meaning having the same.Although similar or equivalent any method and material can also with method described herein and material
To use in implementation or test of the invention, but preferred method and material will now be described.
I. term
Heretofore described " water " word, deionized water, distilled water are referred in the case where being not particularly illustrated or limiting
Or ultrapure water.
II. embodiment
As previously mentioned, at present except use glutamine transaminage can be with cross-linked lipase crude enzyme liquid in addition to, remaining lipase without
Method crosslinking, can not be especially crosslinked with the pure enzyme after ni-sepharose purification at all.Meanwhile enzyme activity loss is obvious after enzyme crosslinking.Mirror
In this, the present inventor to use glutamine transaminage realize protein-crosslinking have conducted extensive research.
The present inventor is the study found that using the peptide chain label for having specific amino acids, by the deoxyribose of the peptide chain label
Nucleotide sequence, which is inserted into protein gene segment albumen obtained with the peptide chain label after expressing, can utilize glutamy
Amine transaminase realizes that protein is orderly crosslinked, while can guarantee the activity of protein without significantly sacrificing.The present invention is based on
State what discovery was made.
For this purpose, for realizing the peptide chain label of protein cross, peptide chain length 8 involved in first aspect present invention
To 15 amino acid, at least provided with a lysine and/or glutamine in peptide chain, amino acid sequence includes Sequence
At least one of No.1-4, as shown in table 1.
Table 1
| Sequence number | Peptide chain label | Peptide chain label amino acid sequence |
| 1 | S peptide | N-KETAAAKFERQHMDS-C |
| 2 | F peptide | N-DYKDDDDG-C |
| 3 | M peptide | N-EQGLISEEDL-C |
| 4 | K2 peptide | N-GKGGGGKGGG-C |
The present inventor the study found that be inserted into the deoxyribonucleotide sequence of above-mentioned peptide chain in protein gene segment,
And make the site of lysine and/or glutamine close to 5 ' ends, thus to obtain the deoxyribonucleotide for having peptide chain label
The protein gene segment of sequence；Using Escherichia coli as host cell, expression has the deoxyribonucleotide sequence of peptide chain label
The protein gene segment of column, so that protein N terminal has peptide chain label, using glutamine transaminage (TGase) to bad ammonia
The function that the primary amine group of acid and the amide group of glutamine have specific recognition to be crosslinked, realizes the albumen for having peptide chain label
The specific recognition of matter is crosslinked.It is therefore contemplated that above-mentioned peptide chain label is the basis realizing protein and capable of being crosslinked by TGase,
And " in the peptide chain, the site of lysine and/or glutamine is close to N-terminal." it may be considered that being to enable protein quilt
The necessary condition of TGase crosslinking.
Correspondingly, the deoxyribonucleotide sequence of the peptide chain includes at least one of Sequence No.5-8, such as
Shown in table 2.
Table 2
According to certain embodiments of the present invention, the method packet of protein cross is realized involved in second aspect of the present invention
It includes:
The deoxyribonucleotide sequence insertion protein gene segment 5 ' of peptide chain label is held, obtains and have peptide by step L
The protein gene segment of the deoxyribonucleotide sequence of chain label；
Step M, the protein gene segment of deoxyribonucleotide sequence of the expression with peptide chain label, obtains and has peptide
The protein of chain label；
Glutamine transaminage (TGase) is added to the substrate solution containing the protein with peptide chain label by step N
In, cross-linking reaction is carried out, cross-linked proteins are made；
Wherein, the peptide chain label is peptide chain label described in first aspect present invention.
The present inventor characterizes albumen obtained by SDS-PAGE, and the molecular weight of protein becomes larger as the result is shown, it was demonstrated that
Protein formed covalent bond, that is, belong to TGase cross-linked proteins formed isopeptide bond as a result, thus proving in the step in paddy ammonia
Crosslinking is carried out to the protein with peptide chain label under amide transaminase (TGase) existence condition and obtains cross-linked proteins；
The present inventor has found that the dosage of enzyme influences the crosslinking rate of albumen, and enzyme amount is very few, then is crosslinked speed by experimental study
It spends slowly, but enzyme amount is excessive, then cost is excessively high, it is preferable that the use of glutamine transaminage in terms of the volume of substrate solution
Amount is 1wt%-3wt%.
In the present invention, the substrate solution is formed by being dissolved in buffer solution containing the protein with peptide chain label, the bottom of with
The content of protein of the object liquor capacity meter with peptide chain label is 5-10mg/mL.
In some preferred embodiments, the buffer solution is the Tris-HCl solution of 0.05mol/L.
In above-mentioned steps N, the temperature of the crosslinking is 20-30 DEG C, and crosslinking time is 4-12 hours.
The side of the protein gene segment of deoxyribonucleotide sequence of peptide chain label is had in the present invention for expressing
Method is not particularly limited, and can be adopted and be expressed the deoxyribonucleotide sequence with peptide chain label with the conventional methods in the field
The protein gene segment of column.For example, in some embodiments: the step M includes:
The genetic fragment of the protein of deoxyribonucleotide sequence with peptide chain label is connected to by step B
Recombinant expression carrier is constructed on pET32b (+) carrier；
Step C, recombinant expression carrier are transferred to the e. coli bl21 (DE3) as host cell, obtain expression bacterial strain；
Step D will carry out fermented and cultured in expression bacterial strain access LB culture medium or TB culture medium, when OD600 reaches 0.8-
When 1.0, inducer IPTG is added and carries out inducing expression culture, suspends again after thallus is then broken born of the same parents, is centrifuged, collects supernatant
It is isolated and purified, obtains the protein sterling for having peptide chain label, wherein the additional amount of the inducer is fermentating liquid volume
0.05wt%.
In above-mentioned steps D, fermented and cultured is carried out in shaking table, the shaking speed is 180rpm；The fermented and cultured
Temperature be 37 DEG C.
In above-mentioned steps D, inducing expression culture is carried out in shaking table, the shaking speed is 180rpm；The induction
The temperature of expression culture is 15-25 DEG C, and the time of the inducing expression culture is 12-20 hours.
The method isolated and purified described in step D is not particularly limited in the present invention, for example, can adopt affine pure
Change method isolates and purifies the albumen in product.
In some preferred embodiments of the invention, affinity purification is carried out using nickel column, and during affinity purification,
Foreign protein is removed using the Tris-HCl solution of 0.05mol/L, is eluted using 0.1mol/L imidazole solution.It is obtained in this way
Albumen to isolate and purify efficiency higher.
In some specific preferred embodiments of the invention, it is crosslinked by glutamine transaminage with peptide chain label
Enhancing albumen, the specific method is as follows:
(1) building of recombinant expression carrier
The genetic fragment of the protein of deoxyribonucleotide sequence with peptide chain label is connected to pET32b (+) to carry
Recombinant expression carrier is constructed on body；
(2) recombinant bacterial strain is constructed
Recombinant expression carrier is transferred to Escherichia coli, obtains expression bacterial strain；
(3) expression of peptide chain label protein matter is had
Expression bacterial strain is subjected to fermented and cultured, carries out inducing expression by inducer of IPTG.It is hanged again after thallus is broken born of the same parents
Floating, centrifugation collects supernatant and carries out affinity purification, obtains the protein for having peptide chain label.
In above-mentioned steps (3), the method for fermented and cultured are as follows: by expression bacterial strain in LB culture medium, 37 DEG C, 180rpm shaking table
Culture, to OD600When reaching 0.8, IPTG is added, temperature is set as 15-25 DEG C, and 180rpm shaking table culture continuously cultivates 12-20
Hour.The additional amount of IPTG is the 0.05% of fermentating liquid volume.
Further, affinity purification obtains pure protein using the destination protein in nickel column affinity purification supernatant；Nickel column parent
When with purifying, foreign protein is removed using the Tris-HCl solution of 0.05mol/L, is eluted using 0.1mol/L imidazole solution.
Further, to peptide chain label protein specific identify crosslinking, glutamine transaminage it is opposite
It is 1%-3% in the usage amount of the total volume of the substrate solution containing the albumen with peptide chain label, crosslinking temperature is 20-30 DEG C,
Crosslinking time is 4-12 hours.
Particularly preferably, the substrate solution is formed by being dissolved in buffer solution containing the protein with peptide chain label, with
The content of protein of the substrate solution stereometer with peptide chain label is 5-10mg/mL.
Further particularly preferably, the buffer solution is the Tris-HCl solution of 0.05mol/L.
The present invention relates to the methods of a kind of method for using genetic engineering and a kind of crosslinking of enzyme process, pass through Bacillus coli expression
Protein with peptide chain label recycles glutamine transaminage (TGase) to carry out the specific recognition of amino acid in peptide chain
The method for realizing protein cross.
Present invention firstly provides the peptide chain labels for having specific amino acids, and the amino acid sequence of the peptide chain label is such as
Shown in table 1, the deoxyribonucleotide sequence of the peptide chain label is as shown in table 2.Secondly, the present invention provides one kind to be based on
The method that protein cross is realized in amino acid specificities identification.This method is using Escherichia coli as host cell, and expression is with upper
The protein gene segment of the deoxyribonucleotide sequence of specific amino acids peptide chain label is stated, obtains and has above-mentioned specific amino
The protein of sour peptide chain label；Affinity purification is carried out to destination protein using nickel column；It is right using glutamine transaminage (TGase)
The function that the primary amine group of lysine and the amide group of glutamine have specific recognition to be crosslinked is realized with peptide chain label
The specific recognition of protein is crosslinked.The present invention identifies amino acid specificities using glutamine transaminage (TGase) and realizes
The method of protein cross will provide basis for protein transformation, the transformation of enzyme molecule performance etc..
III, embodiment
The present invention is specifically described below by way of specific embodiment.Experimental method described below, such as without special theory
It is bright, it is laboratory conventional method.Experimental material described below can be obtained unless otherwise instructed by commercial channel.
Embodiment 1: glutamine transaminage crosslinking has the enhanced green fluorescence protein of S peptide tag
(1) building of recombinant expression carrier
The genetic fragment of the enhanced green fluorescence protein of deoxyribonucleotide sequence with S peptide tag is connected to
Recombinant expression carrier is constructed on pET32b (+) carrier；
(2) recombinant bacterial strain is constructed
Recombinant expression carrier is transferred to Escherichia coli, obtains expression bacterial strain；
(3) expression of peptide chain label protein matter is had
Expression bacterial strain is subjected to fermented and cultured, carries out inducing expression by inducer of IPTG.Expression bacterial strain is cultivated in LB
In base, 37 DEG C, 180rpm shaking table culture, to OD600When reaching 0.8, IPTG is added, temperature is set as 25 DEG C, the training of 180rpm shaking table
It supports, continuous culture 12 hours.The additional amount of IPTG is the 0.05% of fermentating liquid volume.
It suspends, is centrifuged again after thallus is broken born of the same parents, collect supernatant and carry out affinity purification, obtain the increasing for having S peptide tag
Strong type green fluorescent protein.When nickel column affinity purification, foreign protein is removed using the Tris-HCl solution of 0.05mol/L, is used
0.1mol/L imidazole solution is eluted.
Specificity is carried out to the enhanced green fluorescence protein with S peptide tag after purification using glutamine transaminage
Identification crosslinking.Glutamine transaminage is total relative to the substrate solution containing the enhanced green fluorescence protein with S peptide tag
The usage amount of volume is 1wt%, and crosslinking temperature is 20 DEG C, and crosslinking time is 12 hours.
Fig. 1's the results show that swimming lane 1 be glutamine transaminage, swimming lane 2 be enhanced green fluorescence protein, 3 He of swimming lane
4 be the product after crosslinking, is brought and is seen according to item newly-generated in swimming lane 3 and 4, forms the dimer of enhanced green fluorescence protein
And the tetramer, illustrate that enhanced green fluorescence protein is inserted into S peptide tag, can successfully be crosslinked.By to sample, reaction before reacting
The carry out color contrast of the sample of 5 hours samples, the sample for reacting 10 hours and reaction 24 hours is crosslinked the results show that working as
After 10 hours, the fluorescence of enhanced green fluorescence protein starts to disappear, and illustrates that crosslinking is excessive.In conjunction with 1 (A) and 1 (B), when strong
Type green fluorescent protein forms dimer and the tetramer, is still able to maintain fluorescence presence, does not influence the structure of protein itself at this time.
Embodiment 2: glutamine transaminage crosslinking has the enhanced green fluorescence protein of F peptide and M peptide tag
(1) building of recombinant expression carrier
By the enhancing of the deoxyribonucleotide sequence of deoxyribonucleotide sequence and M peptide tag with F peptide tag
The genetic fragment of type green fluorescent protein is connected respectively on pET32b (+) carrier and constructs recombinant expression carrier；
(2) recombinant bacterial strain is constructed
Recombinant expression carrier is transferred to Escherichia coli, obtains expression bacterial strain；
(3) expression of peptide chain label protein matter is had
Expression bacterial strain is subjected to fermented and cultured, carries out inducing expression by inducer of IPTG.Expression bacterial strain is cultivated in LB
In base, 37 DEG C, 180rpm shaking table culture, to OD600When reaching 0.8, IPTG is added, temperature is set as 25 DEG C, the training of 180rpm shaking table
It supports, continuous culture 12 hours.The additional amount of IPTG is the 0.05% of fermentating liquid volume.
It suspends, is centrifuged again after thallus is broken born of the same parents, collect supernatant and carry out affinity purification, obtain have F peptide tag respectively
With the enhanced green fluorescence protein of M peptide tag.When nickel column affinity purification, removed using the Tris-HCl solution of 0.05mol/L
Foreign protein is eluted using 0.1mol/L imidazole solution.
The enhanced green fluorescence protein with F peptide and M peptide after purification is carried out using glutamine transaminage special
Property identification crosslinking.Glutamine transaminage is relative to the substrate solution containing the enhanced green fluorescence protein with F peptide tag
The usage amount of total volume is 3wt%, and crosslinking temperature is 30 DEG C, and crosslinking time is 12 hours.
Swimming lane 1,2,3 is the enhanced green fluorescence protein with F peptide tag after purification, swimming lane 4,5,6 in Fig. 2 (A)
For the enhanced green fluorescence protein with M peptide tag after purification.
In Fig. 2 (B), swimming lane 1,2,3 is that glutamine transaminage is crosslinked F peptide tag and the enhanced green of M peptide tag is glimmering
Photoprotein 6 hours as a result, swimming lane 4,5,6 be glutamine transaminage be crosslinked F peptide tag and M peptide tag enhanced green it is glimmering
12 hours results of photoprotein.
From the above it is found that the high-molecular-weight protein formed is dimer, that is, have the increasing of F peptide tag and M peptide tag
Strong type green fluorescent protein forms the cross-linking result of " one-to-one ".
The sample of sample, reaction 4 hours before comparison reaction, the sample for reacting 8 hours and the sample after reaction 12 hours.
The result shows that: when strong type green fluorescent protein forms dimer, the enhanced green fluorescence egg with F peptide tag and M peptide tag
Leukorrhea does not influence the structure of protein itself with the presence of fluorescence is still able to maintain at this time.
Embodiment 3: the hydrogenlyase of carbonic anhydrase and M peptide tag of the glutamine transaminage crosslinking with F peptide tag
(1) building of recombinant expression carrier
By the deoxyribonucleotide of the carbonic anhydrase of the deoxyribonucleotide sequence with F peptide tag and M peptide tag
The genetic fragment of the hydrogenlyase of sequence is connected respectively on pET32b (+) carrier and constructs recombinant expression carrier；
(2) recombinant bacterial strain is constructed
Recombinant expression carrier is transferred to Escherichia coli, obtains expression bacterial strain；
(3) expression of peptide chain label protein matter is had
Expression bacterial strain is subjected to fermented and cultured, carries out inducing expression by inducer of IPTG.By the expression bacterium of carbonic anhydrase
Strain is in LB culture medium, and 37 DEG C, 180rpm shaking table culture, to OD600When reaching 0.8, IPTG is added, temperature is set as 25 DEG C,
180rpm shaking table culture, continuous culture 20 hours.The additional amount of IPTG is the 0.05% of fermentating liquid volume.By hydrogenlyase
Bacterial strain is expressed in LB culture medium, 37 DEG C, 180rpm shaking table culture, to OD600When reaching 0.8, IPTG is added, temperature is set as
20 DEG C, 180rpm shaking table culture, continuous culture 16 hours.The additional amount of IPTG is the 0.05% of fermentating liquid volume.
It suspends, is centrifuged again after thallus is broken born of the same parents, collect supernatant and carry out affinity purification, obtain have F peptide tag respectively
Carbonic anhydrase and M peptide tag hydrogenlyase.When nickel column affinity purification, gone using the Tris-HCl solution of 0.05mol/L
Except foreign protein, eluted using 0.1mol/L imidazole solution.
Using glutamine transaminage to the formate dehydrogenase of the carbonic anhydrase and M peptide tag with F peptide tag after purification
Enzyme carries out specific recognition crosslinking.Glutamine transaminage is relative to the substrate solution containing the hydrogenlyase with M peptide tag
Total volume usage amount be 2wt%, crosslinking temperature be 25 DEG C, crosslinking time be 4 hours.
Fig. 3's the results show that swimming lane 1 be glutamine transaminage, swimming lane 2 be carbonic anhydrase and M peptide with F peptide tag
The mixture of the hydrogenlyase of label, swimming lane 3 are the product for being crosslinked 0 hour, and swimming lane 4 and 5 is respectively crosslinking 2 hours and 4 is small
When product.The new counterband tape generated in swimming lane 3,4, molecular weight is about 120KDa.According to band show as a result, should be
Carbonic anhydrase (CA) and hydrogenlyase (FDH) form the crosslinking of " one-to-one ".
Fig. 4's the results show that by crosslinking after carbonic anhydrase enzyme activity and hydrogenlyase enzyme activity with it is mixed
The enzyme activity of double enzymes is without being substantially reduced.Show that cross-linking process is little to the loss of enzyme activity.
Embodiment 4: the hydrogenlyase of carbonic anhydrase and M peptide tag of the glutamine transaminage crosslinking with K2 peptide tag
(1) building of recombinant expression carrier
By the deoxyribonucleotide of the carbonic anhydrase of the deoxyribonucleotide sequence with K2 peptide tag and M peptide tag
The genetic fragment of the hydrogenlyase of sequence is connected respectively on pET32b (+) carrier and constructs recombinant expression carrier；
(2) recombinant bacterial strain is constructed
Recombinant expression carrier is transferred to Escherichia coli, obtains expression bacterial strain；
(3) expression of peptide chain label protein matter is had
Expression bacterial strain is subjected to fermented and cultured, carries out inducing expression by inducer of IPTG.By the expression bacterium of carbonic anhydrase
Strain is in LB culture medium, and 37 DEG C, 180rpm shaking table culture, to OD600When reaching 0.8, IPTG is added, temperature is set as 25 DEG C,
180rpm shaking table culture, continuous culture 20 hours.The additional amount of IPTG is the 0.05% of fermentating liquid volume.By hydrogenlyase
Bacterial strain is expressed in LB culture medium, 37 DEG C, 180rpm shaking table culture, to OD600When reaching 0.8, IPTG is added, temperature is set as
20 DEG C, 180rpm shaking table culture, continuous culture 16 hours.The additional amount of IPTG is the 0.05% of fermentating liquid volume.
It suspends, is centrifuged again after thallus is broken born of the same parents, collect supernatant and carry out affinity purification, obtain have K2 peptide tag respectively
Carbonic anhydrase and M peptide tag hydrogenlyase.When nickel column affinity purification, gone using the Tris-HCl solution of 0.05mol/L
Except foreign protein, eluted using 0.1mol/L imidazole solution.
Using glutamine transaminage to the formate dehydrogenase of the carbonic anhydrase and M peptide tag with K2 peptide tag after purification
Enzyme carries out specific recognition crosslinking.Glutamine transaminage is relative to the substrate solution containing the hydrogenlyase with M peptide tag
Total volume usage amount be 2wt%, crosslinking temperature be 25 DEG C, crosslinking time be 12 hours.
Fig. 5's the results show that swimming lane 1 be glutamine transaminage, swimming lane 2 be crosslinking 10 minutes as a result, swimming lane 3 is friendship
0.5 hour product of connection, swimming lane 4,5,6,7,8 are respectively the result for being crosslinked 1 hour, 4 hours, 6 hours, 8 hours and 12 hours.
The display of swimming lane 2 generates the band that molecular weight is about 120KDa, it should for carbonic anhydrase (CA) and hydrogenlyase (FDH) formation
The crosslinking of " one-to-one ".The new counterband tape generated in swimming lane 5, molecular weight is about 200KDa.According to band show as a result, answering
It should be the crosslinking of carbonic anhydrase (CA) and hydrogenlyase (FDH) formation " a pair two ".
Fig. 6's the results show that by crosslinking after carbonic anhydrase enzyme activity and hydrogenlyase enzyme activity with it is mixed
The enzyme activity of double enzymes is without being substantially reduced.Show that cross-linking process is little to the loss of enzyme activity.
It should be noted that embodiment described above for explaining only the invention, is not constituted to of the invention any
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word used in it is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to
And specific method, material and embodiment, it is not intended that the present invention is limited to particular case disclosed in it, on the contrary, this hair
It is bright to can be extended to other all methods and applications with the same function.
Sequence table
<110>Beijing University of Chemical Technology
<120>a kind of that the method for realizing protein cross is identified based on amino acid specificities
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213>(S peptide)
<400> 1
Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
1 5 10 15
<210> 2
<211> 8
<212> PRT
<213>(F peptide)
<400> 2
Asp Tyr Lys Asp Asp Asp Asp Gly
1 5
<210> 3
<211> 10
<212> PRT
<213>(M peptide)
<400> 3
Glu Gln Gly Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 4
<211> 10
<212> PRT
<213>(K2 peptide)
<400> 4
Gly Lys Gly Gly Gly Gly Lys Gly Gly Gly
1 5 10
<210> 5
<211> 45
<212> DNA
<213>(S peptide)
<400> 5
aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg acagc 45
<210> 6
<211> 24
<212> DNA
<213>(F peptide)
<400> 6
gattacaagg atgacgacga tggt 24
<210> 7
<211> 30
<212> DNA
<213>(M peptide)
<400> 7
gagcagggtc tcatctctga agaggatctg 30
<210> 8
<211> 30
<212> DNA
<213>(K2 peptide)
<400> 8
ggtaaaggtg gtggtggtaa aggtggtggt 30
Claims (10)
1. a kind of peptide chain label for realizing protein cross, peptide chain length is 8 to 15 amino acid, at least band in peptide chain
There are a lysine and/or glutamine, amino acid sequence includes at least one of Sequence No.1-4.
2. peptide chain label according to claim 1, which is characterized in that in the peptide chain, lysine and/or glutamine
Site close to N-terminal.
3. peptide chain label according to claim 1 or 2, which is characterized in that the deoxyribonucleotide sequence of the peptide chain
Including at least one of Sequence No.5-8.
4. a kind of method for realizing protein cross comprising:
The deoxyribonucleotide sequence insertion protein gene segment 5 ' of peptide chain label is held, obtains and have peptide chain mark by step L
The protein gene segment of the deoxyribonucleotide sequence of label；
Step M, the protein gene segment of deoxyribonucleotide sequence of the expression with peptide chain label, obtains and has peptide chain mark
The protein of label；
Step N carries out crosslinking to the protein with peptide chain label under glutamine transaminage (TGase) existence condition and is made
Crosslinking protein；
Wherein, the peptide chain label is the peptide chain label as described in any one of claim 1-3.
5. according to the method described in claim 4, it is characterized in that, glutamine transaminage is added to containing band in step N
In the substrate solution for having the protein of peptide chain label, cross-linking reaction is carried out, crosslinking protein is made；Preferably, with substrate solution
The usage amount of stereometer glutamine transaminage is 1wt%-3wt%；And/or the substrate solution is by containing with peptide chain label
Protein be dissolved in buffer solution and being formed, the content containing the protein with peptide chain label in terms of substrate solution volume is 5-
10mg/mL；It is further preferred that the buffer solution is the Tris-HCl solution of 0.05mol/L.
6. method according to claim 4 or 5, which is characterized in that in step N, the temperature of the crosslinking is 20-30
℃；And/or crosslinking time is 4-12 hours.
7. method according to any one of claims 4 to 6, which is characterized in that the step M includes:
The genetic fragment of the protein of deoxyribonucleotide sequence with peptide chain label is connected to structure on carrier by step B
Build recombinant expression carrier；
Step C, recombinant expression carrier are transferred to host cell, obtain expression bacterial strain；
Expression bacterial strain is carried out fermented and cultured, and carries out inducing expression with inducer by step D, then breaks thallus after born of the same parents again
It suspends, centrifugation is collected supernatant and isolated and purified, obtains the protein sterling for having peptide chain label.
8. the method according to the description of claim 7 is characterized in that the host cell is Escherichia coli, the preferably described host
Cell is e. coli bl21 (DE3)；And/or the carrier is pET32b (+) carrier；And/or the inducer is IPTG,
Preferably, the additional amount of the IPTG is the 0.05% of fermentating liquid volume.
9. method according to claim 7 or 8, which is characterized in that in step D, will be in expression bacterial strain access culture medium
Fermented and cultured is carried out, inducer is then added and carries out inducing expression culture；Preferably, it will be carried out in expression bacterial strain access culture medium
Fermented and cultured is added inducer and carries out inducing expression culture when OD600 reaches 0.8-1.0；And/or the culture medium is LB
Culture medium or TB culture medium；And/or the temperature of fermented and cultured is 37 DEG C；And/or the temperature of inducing expression culture is 15-25
℃；It is further preferred that carrying out fermented and cultured in shaking table；Preferably, the shaking speed is 180rpm；And/or in shaking table
Middle progress inducing expression culture；Preferably, the shaking speed is 180rpm；And/or the time of the inducing expression culture is
12-20 hours.
10. the method according to any one of claim 6-8, which is characterized in that in step D, described to isolate and purify
Method include affinity purification；Preferably, affinity purification is carried out using nickel column；It is further preferred that during affinity purification,
Foreign protein is removed using the Tris-HCl solution of 0.05mol/L, is eluted using 0.1mol/L imidazole solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110835364A (en) * | 2019-11-29 | 2020-02-25 | 北京化工大学 | Method for realizing protein separation and purification based on amino acid specificity identification |
| CN111138513A (en) * | 2020-01-06 | 2020-05-12 | 天津科技大学 | Screening of novel transglutaminase Cross-Linked peptides |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421199A (en) * | 2013-05-09 | 2013-12-04 | 河南工业大学 | Gamma-polyglutamic acid hydrogel obtained by using enzymic method, and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103421199A (en) * | 2013-05-09 | 2013-12-04 | 河南工业大学 | Gamma-polyglutamic acid hydrogel obtained by using enzymic method, and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| KAMIYA N等: "S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis", 《BIOCONJUGATE CHEMISTRY》 * |
| STROP P: "Versatility of Microbial Transglutaminase", 《BIOCONJUGATE CHEMISTRY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110835364A (en) * | 2019-11-29 | 2020-02-25 | 北京化工大学 | Method for realizing protein separation and purification based on amino acid specificity identification |
| CN110835364B (en) * | 2019-11-29 | 2021-10-15 | 北京化工大学 | Method for realizing protein separation and purification based on amino acid specificity identification |
| CN111138513A (en) * | 2020-01-06 | 2020-05-12 | 天津科技大学 | Screening of novel transglutaminase Cross-Linked peptides |
| CN111138513B (en) * | 2020-01-06 | 2022-10-18 | 天津科技大学 | Screening of Cross-Linked peptides of Glutamine transaminase |
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