CN103060283B - Polypeptide for inhibiting activity of bacillus subtilis transglutaminase (BTG) and its screening method and use - Google Patents

Abstract

The invention relates to a polypeptide for inhibiting an activity of bacillus subtilis transglutaminase (BTG) and its screening method and use. The polypeptide has an amino acid sequence shown in the formula of SEQ ID No: 1. The polypeptide provided by the invention can inhibit the activity of BTG by a fusion protein and has an inhibition rate more than 60%. Through the polypeptide, the activity of expressed BTG having strong cytotoxicity is inhibited in a host cell and then is recovered after the inhibitor is removed under the action of a blood coagulation factor Xa.

Description

A kind of polypeptide and screening method and application that suppresses subtilis transglutaminase activity

Technical field

The invention belongs to biological technical field, especially a kind of polypeptide and screening method and application that suppresses subtilis Transglutaminase EC2.3.2.13 (subtilis Transglutaminase EC2.3.2.13 is called for short BTG) activity.

Background technology

Transglutaminase EC2.3.2.13 (transglutaminase; EC2.3.2.13; be called for short TG); it is a kind of enzyme of catalyzing acyl shift reaction; can catalytic proteins form ε-(γ-Glu)-Lys isopeptide bond between interior, the intermolecular and protein and amino acids of molecule and make its covalent cross-linking polymerization; thereby directly change structure and the function of protein itself and protein accompanying cell, tissue etc., thereby this enzyme has a wide range of applications in foodstuffs industry and medicine industry.The production method of TG has two kinds of animal vegetable tissue extraction method and microbe fermentation methods.The early stage production of TG adopts animal tissues's extraction method, but due to animal tissues source less, extraction process complexity, the rate of recovery is low, product cost is too expensive cause, the production application of enzyme is very limited always.Microbe-derived TG does not rely on Ca because of its enzymic activity ²⁺, and be hopeful to realize industrial production most, and attracting wide attention, the current only TG of luxuriant source Streptomyces has realized commercially producing and has been applied in food-processing.The TG in streptomyces source has signal peptide and propetide, due to the effect of propetide, makes the TG of Streptomyces in cell, there is no activity, and does not damage cell, is then secreted into extracellular by signal peptide, realizes the production of TG.

Compare with the TG of Streptomyces; much less is wanted in the TG research in subtilis source; within 1996, just in subtilis, find first TG by Kobayashi etc.; it does not have signal peptide and propetide; only be present on gemma; crosslinked spore surface albumen, prevents that it is by proteasome degradation, thereby plays the effect of protection gemma.Because TG has the activity of protein-crosslinking, host cell is had to toxic action, therefore, although Portugal Placido in 2007 etc. realize BTG at expression in escherichia coli, under different inductions and culture condition, enzyme yield is all very low.At present, be still difficult to prepare in a large number the BTG in subtilis source.

By retrieval, not yet find the patent publication us relevant to patent application of the present invention:

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of polypeptide and screening method and application that suppresses subtilis transglutaminase activity is provided, this polypeptide can be expressed at host cell low activity BTG, alleviate the toxic action of BTG to host cell, and its activity can be restored after proteolytic enzyme simple process, for the high efficient expression of BTG is laid a good foundation.

The technical scheme that the present invention takes is:

Aminoacid sequence is that the polypeptide of SEQIDNo:11 is in the application suppressing in subtilis transglutaminase activity.

And the method steps of application is as follows:

Described polypeptide and factor Xa can be identified to restriction enzyme site sequence to be connected, form polypeptide-I-E-G-R, subtilis Transglutaminase EC2.3.2.13 gene reorientation is cloned into the downstream of polypeptide-I-E-G-R, forms polypeptide-I-E-G-R-BTG fragment; This polypeptide suppresses the activity of BTG with the form of fusion rotein, remove this polypeptide after factor Xa enzyme is cut, and recovers subtilis transglutaminase activity.

Suppress the polypeptide of subtilis transglutaminase activity, described polypeptide is through the replacement of one or several amino-acid residue and/or the polypeptide of disappearance and/or interpolation and inhibition subtilis transglutaminase activity by the amino acid residue sequence of SEQIDNo:1.

Screen a method that suppresses subtilis transglutaminase activity polypeptide, step is as follows:

(1) select the Transglutaminase EC2.3.2.13 propeptide sequence of the different streptomycete in source;

(2) the N end that by overlapping extension PCR, the different streptomycete Transglutaminase EC2.3.2.13 propetide in source is cloned into subtilis Transglutaminase EC2.3.2.13, adds factor Xa recognition sequence between the two and connects;

(3) the NdeI and the HindIII enzyme that whole encoding sequences are cloned into pET-28a (+) are cut between recognition site;

(4) at E.ColiBL21(DE3) recombinant expressed in bacterial strain, affinitive layer purification obtains the fusion rotein of the encoding sequence that HIS merges;

(5) live by the enzyme of Fluorometric assay fusion rotein, screening, to the highest propeptide sequence of subtilis transglutaminase activity inhibiting rate, must suppress the polypeptide of subtilis transglutaminase activity.

And the described step (1) different streptomycete in middle source is S.cinnamoneus, S.caniferus, S.fradiae, S.hygroscopicus, S.mobaraense, S.netropsis and S.platensis.

And, described step (5) in the concrete steps of Fluorometric assay as follows:

Protein liquid after purifying is concentrated is respectively got 500 μ L, be divided equally into two parts, portion is wherein cut 6h with factor Xa enzyme, the ripe subtilis Transglutaminase EC2.3.2.13 of N end peptide sequence has been removed in acquisition, record the fluorescence intensity of each protein liquid, calculate enzyme and live, live with respect to the enzyme after cutting by the enzyme work of relatively cutting without factor Xa enzyme, obtain the inhibiting rate of each peptide sequence to subtilis transglutaminase activity.

And the detecting step of described fluorescence intensity is:

The cumulative volume 1mL:N of reaction system, N-dimethyl casein 0.67g/L, red sulphur cadaverine (MDC) 8.33 μ mol/L, TrisHClpH7.550mmol/L, DTT3mmol/L, enzyme liquid 200 μ L; Reaction system, in 37 ℃ of reaction 30min, adds 10mmol/L (NH ₄) ₂sO ₄termination reaction, fluorescence intensity under excitation wavelength 350nm and emission wavelength 500nm.

Advantage of the present invention and positively effect are:

1, the polypeptide that the present invention suppresses subtilis transglutaminase activity is by the activity of the form inhibition BTG of fusion rotein, inhibiting rate can reach more than 60%, can make the BTG that cytotoxicity is stronger first express in host cell with the downtrod form of activity, then under the effect of factor Xa (FactorXa), remove inhibition, make again its activity be restored.

2, polypeptide of the present invention is owing to can specificity suppressing the activity of BTG, and can remove at any time, derepression effect, can be applied to and suppress in subtilis transglutaminase activity, and is applied in the Transglutaminase EC2.3.2.13 in preparation subtilis source.

3, the screening method of polypeptide of the present invention is as the TG of Streptomyces, add the preceding paragraph polypeptide at the N of BTG end, first suppress the activity of BTG by polypeptide, its low activity is expressed, then by the cutting of proteolytic enzyme, remove polypeptide, remove the inhibition to BTG, give full play to its biological activity, thereby alleviate its toxic action to host cell, for its high efficient expression that can realize in host cell lays the foundation.

Accompanying drawing explanation

Fig. 1 is concentrated figure after the purifying of five kinds of fusion roteins of the present invention, and swimming lane 1-5 is followed successively by his-proCan-I-E-G-R-BTG, his-proFra-I-E-G-R-BTG, his-proHyg-I-E-G-R-BTG, his-proNet-I-E-G-R-BTG, his-proPla-I-E-G-R-BTG;

Fig. 2 is concentrated figure after the purifying of the another two kinds of fusion roteins of the present invention, and swimming lane 1-2 is followed successively by his-proMob-I-E-G-R-BTG, his-proCin-I-E-G-R-BTG;

Fig. 3 is the inhibition figures of seven kinds of fusion roteins of the present invention to BTG activity.

Embodiment

Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.

Method in following embodiment, if no special instructions, is ordinary method.

The TG in streptomyces source has signal peptide and propetide, due to the effect of propetide, makes the TG of Streptomyces in cell, there is no activity, cannot play the effect of crosslinking protein, thereby not damage cell.But the BTG in subtilis source does not have signal peptide and propetide, only has the activated mature peptide of tool, therefore wishes to suppress by the propetide of streptomyces TG the activity of BTG.

Select to derive from 7 kinds of different streptomycete (S.cinnamoneus, S.caniferus, S.fradiae, S.hygroscopicus, S.mobaraense, S.netropsis, S.platensis) TG propeptide sequences from having compared, be designated as respectively proCin, proCan, proFra, proHyg, proMob, proNet, proPla, be combined with BTG and suppress the situation of BTG.The N end that mainly by overlapping extension PCR, the different streptomycete TG propetide in source is cloned into BTG, adds FactorXa recognition sequence (ATCGAGGGAAGG) between the two and connects.The NdeI and the HindIII enzyme that whole encoding sequences are cloned into pET-28a (+) are cut between recognition site again, thereby remove the T7Tag on pET-28a (+) carrier, prevent its interference to polypeptide retarding effect.Recombinant expressed in E.ColiBL21 (DE3) bacterial strain, affinitive layer purification obtains fusion rotein his-proCin-I-E-G-R-BTG, his-proCan-I-E-G-R-BTG, his-proFra-I-E-G-R-BTG, his-proHyg-I-E-G-R-BTG, his-proMob-I-E-G-R-BTG, his-proNet-I-E-G-R-BTG, the his-proPla-I-E-G-R-BTG of 7 kinds of sequences of coding of HIS fusion.

The present invention lives by Fluorometric assay enzyme, and Analysis and Screening has obtained the TG propeptide sequence (sequence 11 in sequence table) that the stronger S.caniferus of restraining effect originates.

One, the design that suppresses the polypeptide of BTG activity is synthesized and screening active ingredients

1, the design of polypeptid coding sequence is synthetic

First design 7 kinds of not encoding sequences of homopolypeptide, represented by A1, A2, A3, A4, A5, A6, A7 respectively, this sequence is followed successively by 7 kinds of TG propeptide sequences (proCin, proCan, proFra, proHyg, proMob, proNet, proPla), the N end 31bp sequence of FactorXa specific recognition sequence and BTG.Synthesized and the NdeI and the HindIII enzyme that are cloned in pUC57 cut between recognition site by biotech firm, plasmid is stored in E.Coli Top10.(gene entrusts Jin Weizhi bio tech ltd, Suzhou to synthesize);

Then utilize the little extraction reagent kit of plasmid to extract plasmid from E.Coli Top10,80 μ L system double digestion 3-4h.

In the following order, each composition is mixed in sterilizing thin wall centrifugal tube,

Finally all through agarose gel electrophoresis and cut glue reclaim obtain aforementioned polypeptides Gene A 1, A2, A3, A4, A5, A6, A7, as the upstream gene of overlapping PCR.

2, the acquisition of BTG gene

The B.subtilis168 preserving from this laboratory, extract genome as template, PCR obtains BTG gene, as the downstream gene B of overlapping PCR.

Upstream primer P1:5 '-ATGATTATTGTATCAGGACAATTGCTCCGT-3 '

Downstream primer P2:5 '-CCCAAGCTTTTAGCGGACGATGCGG-3'

In the following order, each composition is mixed in sterilizing thin wall centrifugal tube,

Amplification condition: 95 ℃ of 5min, 1 circulation; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 60s, 30 circulations; 72 ℃ of 10min, 1 circulation.

Wherein upstream primer P15 ' end is not containing restriction enzyme site, and downstream primer P25 ' end is cut recognition site containing HindIII enzyme.

3, the splicing of gene series connection

Using Gene A 1, A2, A3, A4, A5, A6, A7 and gene B as template, carry out overlapping extension PCR respectively.

Upstream primer F1:5'-CGCCATATGGGCGATGGGGAAGAGA-3'

Upstream primer F2:5 '-CGCCATATGGCCAGCGGCGG-3 '

Upstream primer F3:5 '-CGCCATATGGCCGACAGCGGC-3 '

Upstream primer F4:5 '-CGCCATATGGCTAGCGGTGACGACG-3 '

Upstream primer F5:5 '-CGCCATATGGACAATGGCGCGGG-3 '

Upstream primer F6:5 '-CGCCATATGGCCGACGCCGG-3 '

Upstream primer F7:5 '-CGCCATATGGCCAGCCGCGG-3 '

Downstream primer R1:5 '-CCCAAGCTTTTAGCGGACGATGCGG-3'

Wherein all upstream primer 5 ' ends all contain NdeI restriction enzyme site, and downstream primer 5 ' end is containing HindIII restriction enzyme site.

In the following order, each composition is mixed in sterilizing thin wall centrifugal tube,

（1）PCR1

Amplification condition: 95 ℃ of 5min, 1 circulation; 94 ℃ of 40s, 57.4 ℃ of 40s, 72 ℃ of 60s, 30 circulations; 72 ℃ of 10min, 1 circulation.

Take Gene A 2 and gene B as template, primers F 2 and R1 are primer; Gene A 3 and gene B are template, and primers F 3 and R1 are primer; Gene A 4 and gene B are template, and primers F 4 and R1 are primer; Gene A 6 and gene B are template, and primers F 6 and R1 are primer; Gene A 7 and gene B are template, and primers F 7 and R1 are primer, carry out PCR1 amplification under above-mentioned system and condition.Then through agarose gel electrophoresis and cut glue and reclaim and obtain proCan-I-E-G-R-BTG, proFra-I-E-G-R-BTG, proHyg-I-E-G-R-BTG, proNet-I-E-G-R-BTG, five kinds of genes of proPla-I-E-G-R-BTG.

（2）PCR2

Amplification condition: 95 ℃ of 5min, 1 circulation; 94 ℃ of 40s, 52.6 ℃ of 40s, 72 ℃ of 60s, 30 circulations; 72 ℃ of 10min, 1 circulation.

Take Gene A 1 and gene B as template, primers F 1 and R1 are primer; Gene A 5 and gene B are template, and primers F 5 and R1 are primer, carry out PCR2 amplification under above-mentioned system and condition.Then by agarose gel electrophoresis and cut glue and reclaim and obtain proCin-I-E-G-R-BTG, two kinds of genes of proMob-I-E-G-R-BTG.

4, the structure of recombinant expression vector

(1) by above-mentioned obtained NdeI and HindIII double digestion for 7 kinds of genes, enzyme is cut product through DNA purification kit (this test kit is purchased from border biological gene Science and Technology Ltd. of the village, Beijing ally) purifying.

(2) by pET-28a (+) plasmid with NdeI with after HindIII double digestion, purifying, under the condition of 16 ℃, be connected 12h with (1) step purified product respectively, obtain 7 kinds of recombinant plasmids.

(3) recombinant plasmid of 4 μ L is turned to 40 μ LE.ColiDH5 α competent cells; be coated on containing on the flat board of kantlex (50 μ g/ml); select transformant from flat board; after checking is correct, extract plasmid; change and proceed to E.ColiBL21(DE3 again) in, the positive transformant that checking is correct preserved.

Two, the separation and purification of fusion rotein his-proCin-I-E-G-R-BTG, his-proCan-I-E-G-R-BTG, his-proFra-I-E-G-R-BTG, his-proHyg-I-E-G-R-BTG, his-proMob-I-E-G-R-BTG, his-proNet-I-E-G-R-BTG, his-proPla-I-E-G-R-BTG

By the positive transformant of 37 ℃ of overnight incubation, to transfer in the LB substratum that contains 50 μ g/mL kantlex in 800mL by the inoculum size of 1% (v:v), shaking culture is to OD ₆₀₀be 0.8 o'clock, adding final concentration is the IPTG of 1mmol/L, abduction delivering 16h under 16 ℃, 160r/min condition.Centrifugal collecting cell, high-pressure homogenization smudge cells, through Ni ²⁺-NTAAgarose medium (purchased from GE company) affinity purification, by super filter tube (purchased from Millipore company) concentrated rear (as Fig. 1 and Fig. 2), obtain with histidine-tagged fusion rotein his-proCin-I-E-G-R-BTG, his-proCan-I-E-G-R-BTG, his-proFra-I-E-G-R-BTG, his-proHyg-I-E-G-R-BTG, his-proMob-I-E-G-R-BTG, his-proNet-I-E-G-R-BTG, his-proPla-I-E-G-R-BTG again.

Three, Fluorometric assay proCin, proCan, proFra, proHyg, proMob, proNet, the inhibition situation of proPla to BTG activity.

Protein liquid after purifying is concentrated is respectively got 500 μ L, is divided equally into two parts, and portion is wherein cut 6h with FactorXa enzyme, obtains the ripe BTG that has removed N end peptide sequence.Repeat to test by three times, the enzyme work of relatively cutting without FactorXa enzyme is lived with respect to the enzyme after cutting, and can learn the inhibition situation of peptide sequence to BTG activity.

Concrete grammar is as follows:

Cumulative volume 1mL:N, N-dimethyl casein 0.67g/L, red sulphur cadaverine (MDC) 8.33 μ mol/L, TrisHCl (pH7.5) 50mmol/L, DTT3mmol/L, enzyme liquid 200 μ L.Reaction system, in 37 ℃ of reaction 30min, adds 10mmol/L (NH ₄) ₂sO ₄termination reaction, fluorescence intensity under excitation wavelength 350nm and emission wavelength 500nm.

(2) enzyme is lived and is defined

The MDC of per minute catalysis 1nmol/L is inserted into N, and the required enzyme amount of N-dimethyl casein is defined as a unit of activity.

(3) enzyme work is calculated

Wherein Δ I is the increased value of reaction system fluorescence intensity, i.e. Δ I=I-I ₀, the fluorescence intensity that I is product, I ₀control reaction fluorescence intensity during for enzyme-added liquid not, [MDC] is the concentration of MDC in reaction system.

The fluorescence intensity (I) of product is control reaction fluorescence intensity (I during for enzyme-added liquid not ₀) 13 times, if [MDC] is all inserted into N, in N-dimethyl casein, the fluorescence intensity of system should be 13 × I ₀, measure increased value Δ the I (=I-I of reaction system fluorescence intensity ₀), can obtain the insertion of MDC.

As shown in Figure 3, fusion rotein his-proCan-I-E-G-R-BTG enzyme ratio alive after the work of FactorXa cutting preferment is with respect to cutting is minimum for result, shows that the TG propeptide sequence (sequence 11 in sequence table) in S.caniferus source suppresses the strongest to BTG is active.

After testing, polypeptide of the present invention suppresses the activity of BTG by the form of fusion rotein, and inhibiting rate can reach more than 60%.

Because this polypeptide can specificity suppresses the activity of BTG, and can remove at any time, derepression effect, can be applied to and suppress in subtilis transglutaminase activity, finally lay the foundation at the high efficient expression of host cell for BTG, therefore can be applied in the Transglutaminase EC2.3.2.13 in preparation subtilis source.

The aminoacid sequence of SEQ ID No:1

Claims (6)

1. aminoacid sequence is that the polypeptide of SEQ ID No:11 is in the application suppressing in subtilis transglutaminase activity.

2. application according to claim 1, is characterized in that: the method steps of application is as follows:

Described polypeptide and factor Xa can be identified to restriction enzyme site sequence to be connected, form polypeptide-I-E-G-R, subtilis Transglutaminase EC2.3.2.13 gene reorientation is cloned into the downstream of polypeptide-I-E-G-R, forms polypeptide-I-E-G-R-BTG fragment; This polypeptide suppresses the activity of subtilis Transglutaminase EC2.3.2.13 with the form of fusion rotein, remove this polypeptide after factor Xa enzyme is cut, and recovers subtilis transglutaminase activity.

3. screen a method that suppresses subtilis transglutaminase activity polypeptide, it is characterized in that: step is as follows:

(1) select the Transglutaminase EC2.3.2.13 propeptide sequence of the different streptomycete in source;

(2) the N end that by overlapping extension PCR, the different streptomycete Transglutaminase EC2.3.2.13 propetide in source is cloned into subtilis Transglutaminase EC2.3.2.13, adds factor Xa recognition sequence between the two and connects;

(3) the NdeI and the HindIII enzyme that whole encoding sequences are cloned into pET-28a (+) are cut between recognition site;

(4) at E.ColiBL21(DE3) recombinant expressed in bacterial strain, affinitive layer purification obtains the fusion rotein of the encoding sequence that HIS merges;

(5) live by the enzyme of Fluorometric assay fusion rotein, screening, to the highest propeptide sequence of subtilis transglutaminase activity inhibiting rate, must suppress the polypeptide of subtilis transglutaminase activity.

4. screening according to claim 3 suppresses the method for subtilis transglutaminase activity polypeptide, it is characterized in that: the described step (1) different streptomycete in middle source is Streptomycescinnamoneus, Streptomycescaniferus, Streptomycesfradiae, Streptomyceshygroscopicus, Streptomycesmobaraense, Streptomycesnetropsis and Streptomycesplatensis.

5. screening according to claim 3 suppresses the method for subtilis transglutaminase activity polypeptide, it is characterized in that: described step (5) in the concrete steps of Fluorometric assay as follows:

Protein liquid after purifying is concentrated is respectively got 500 μ L, be divided equally into two parts, portion is wherein cut 6h with factor Xa enzyme, the ripe subtilis Transglutaminase EC2.3.2.13 of N end peptide sequence has been removed in acquisition, record the fluorescence intensity of each protein liquid, calculate enzyme and live, live with respect to the enzyme after cutting by the enzyme work of relatively cutting without factor Xa enzyme, obtain the inhibiting rate of each peptide sequence to subtilis transglutaminase activity.

6. screen according to claim 5 the method that suppresses subtilis transglutaminase activity polypeptide, it is characterized in that: the detecting step of described fluorescence intensity is:

The cumulative volume 1mL:N of reaction system, N-dimethyl casein 0.67g/L, red sulphur cadaverine (MDC) 8.33 μ mol/L, TrisHClpH7.550mmol/L, DTT3mmol/L, enzyme liquid 200 μ L; Reaction system, in 37 ℃ of reaction 30min, adds 10mmol/L (NH ₄) ₂sO ₄termination reaction, fluorescence intensity under excitation wavelength 350nm and emission wavelength 500nm.

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