CN107034202A - A kind of N glycosyl transferases AaNGT and its application - Google Patents
A kind of N glycosyl transferases AaNGT and its application Download PDFInfo
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- CN107034202A CN107034202A CN201710497269.9A CN201710497269A CN107034202A CN 107034202 A CN107034202 A CN 107034202A CN 201710497269 A CN201710497269 A CN 201710497269A CN 107034202 A CN107034202 A CN 107034202A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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Abstract
The invention discloses a kind of N glycosyl transferases AaNGT, its amino acid sequence is as shown in SEQ ID NO.1.The invention also discloses the N glycosyl transferases AaNGT the glycosylation modified aspect of polypeptide application.AaNGT provides a kind of simple and efficient method for the formation of glycopeptide, its the ability using UDP Glc will apparently higher than it has been reported that the N glycosyl transferase ApNGT from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), and AaNGT can also be by special glycosyl donor UDP GlcNH2It is transferred on the polypeptide containing N glycosylation sites, then by using other glycosyl transferases by GlcNH2GlcNAc is changed into, natural N sugar connections are formed, this provides a kind of new approach for the development of glucoprotein vaccine.This helps to make AaNGT turn into a kind of toolenzyme of protein modification.
Description
Technical field
N glycosyls in Haemophilus aphrophilus (Aggregatibacter aphrophilus) are derived from the present invention relates to one kind
Transferase AaNGT and its application, belong to sugar engineering technical field in molecular biology.
Background technology
Glycoprotein is the important physiological activator of a class as formed by oligosaccharides and the link of polypeptide chain covalent modification, and it is wide
It is general to be present in cell membrane, cytoplasm, blood plasma and mucus.It is in cell signal identification, neuromodulation, intercellular information
Very important role is play during reception and registration and immunological regulation.Protein it is glycosylation modified according to importance successively
It is divided into N-O-P-C- and G- glycosylations, wherein N- connections are the most universal, first sugar of N- connections is GlcNAc.Its research is most
To be thorough.Research finds sugar antigens being combined with carrier protein, and the glucoprotein vaccine of formation can stimulate body to produce and be immunized
Remember, therefore the research of glucoprotein vaccine is attracted wide attention.And the acquisition of glycoprotein was by direct in organism in the past
Extract, or carry out chemical synthesis in vitro, both approaches have that step is numerous and diverse, the higher shortcoming of cost.Through retrieval, have
Close the N glycosyl transferases AaNGT and its zymetology derived from Haemophilus aphrophilus (Aggregatibacter aphrophilus)
Property with its to polypeptide carry out N be connected it is glycosylation modified in application yet there are no report.
The content of the invention
In view of the shortcomings of the prior art, Haemophilus aphrophilus (Aggregati is derived from it is an object of the invention to provide one kind
Bacter aphrophilus) in N glycosyl transferases AaNGT and its to polypeptide carry out N connections it is glycosylation modified in application.
N glycosyl transferases AaNGT of the present invention, it is characterised in that：The N glycosyl transferases AaNGT derives from thermophilic foam
Haemophilus (Aggregatibacter aphrophilus), its amino acid sequence is as shown in SEQ ID NO.1.
Above-mentioned N glycosyl transferases AaNGT condenses bacillus (Aggregatibacter aphrophilus) from thermophilic foam
AaNGT genes are responsible for synthesizing by Nanjing Jin Sirui companies and built with carrier pET45b, being lured by e. coli bl21 fermentation
Lead expression, purifying and reclaim acquisition.
N glycosyl transferases AaNGT of the present invention is in the application to polypeptide progress N connections in glycosylation modified.
Wherein, the method for the application is：Using AaNGT as a kind of glycosylation modified toolenzyme of polypeptide, by UDP-Glc
Or UDP-Gal is transferred to the polypeptide of the sequence containing N-X-S/T, glycopeptide is formed；Or AaNGT is by special glycosyl donor UDP-GlcNH2
It is transferred on the polypeptide containing N glycosylation sites, recycles other glycosyl transferases by GlcNH2GlcNAc is changed into, is formed
Natural N sugar connections.
N glycosyl transferases ApNGT disclosed by the invention is higher than deriving from of having reported for work in the efficiency using UDP-Glc
The N glycosyl transferase ApNGT of Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), and AaNGT
Unique glycosyl donor UDP-GlcNH can also be utilized2It is transferred on the polypeptide containing N glycosylation sites, and then passes through other
Glycosyl transferase is by GlcNH2GlcNAc is changed into, the glycopeptide of natural N connections is formed, this is the conjunction of natural N connections glycoprotein
It is that the exploitation of glucoprotein vaccine is laid a good foundation into there is provided a kind of new approach.
Brief description of the drawings
Fig. 1：The agarose gel electrophoresis result of the pET-45b plasmid extractions of the gene containing AaNGT
Wherein：M represents agarose gel electrophoresis Marker, and No. 1 swimming lane is the plasmid extracted from BL21 experimental strains
BL21pET45b-AaNGT, No. 2 swimming lanes are original structure plasmid pET45b-AaNGT as positive control.
Fig. 2：The SDS-PAGE coomassie brilliant blue staining results of AaNGT albumen
Wherein：M represents that albumen Maker, AaNGT molecular weight No. 1 swimming lane about in 70KDa or so, figure is after inducing
Thalline；No. 2 swimming lanes are the supernatant after ultrasonication, and No. 3 swimming lanes are the precipitation after ultrasonication；No. 4 swimming lanes are loading efflux,
5, No. 6 swimming lanes are low concentration imidazoles elution destination protein effluxes；No. 7 swimming lanes are the destination protein liquid of 250mM imidazoles elution.
Fig. 3：HPLC detects AaNGT enzyme activity result figures.
Fig. 4：Simple qualification result figure.
Fig. 5：AaNGT optimum temperature result figures.
Fig. 6：AaNGT optimal pH result figures.
Fig. 7：Metal ion AaNGT enzyme activity influences result figure.
Fig. 8：HPLC detections AaNGT is compared with ApNGT using UDP-Glc abilities.
Fig. 9：HPLC detects AaNGT glycosyl donor substrate specificities.
Figure 10：Reaction product simpleness qualification result figure.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means all in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of the spirit and scope of invention, various variations or change are carried out to the material component in these embodiments and consumption
Fall within protection scope of the present invention.
The AaNGT provided below in conjunction with the accompanying drawings the present invention (derives from Haemophilus aphrophilus Aggregatibacter
) and its concrete application is described in detail aphrophilus.
The peptide substrate that this experiment is used is to carry the hexapeptide that TAMRA is marked in N-terminal by what Nanjing Jin Sirui companies synthesized
DANYTK, reaction solution is handled, reaction is analyzed using HPLC after the reaction of this experiment zymetology, point used
Analysis post is the reverse posts of C18, and the separable programming of use is as shown in table 1-1：
Table 1-1 HPLC analysis programs
The strain and plasmid origin that the present invention is designed are as follows：
Table 1-2 bacterial strain uses therefors of the present invention and plasmid
Embodiment 1：N glycosyl transferases AaNGT preparation
1. express the structure of bacterial strain
The plasmid that Jin Sirui companies are synthesized is transferred in BL21 competent cells, 42 DEG C of thermal shock 1min, applies Amp flat boards.Will
Single bacterium colony is chosen to 5ml 37 DEG C of 200rpm of Tube propagation base and cultivates 12h, upgrading grain checking.Plasmid agarose gel electrophoresis result
See Fig. 1.
The expression and purification of 2.AaNGT albumen
Single bacterium colony BL21pET45b-AaNGT is chosen in 50ml LB culture mediums, (the ampicillin containing 50ug/ml
Amp) 37 DEG C of 200rpm activation 12h.Then expand culture, bacterium solution is transferred in 1L culture mediums (the ammonia benzyl mould containing 50ug/ml
Plain Amp), 37 DEG C of 200rpm is cultivated after about 3.5h, is surveyed OD values, is worked as OD600Be worth for 0.6 when, ice bath 20min adds 400 afterwards
μ L 0.5M IPTG, inducible protein expression (16 DEG C of 200rpm).Induce after 20h, 10000rpm centrifugations 10min collects thalline, uses
Binding Buffer rinse thalline, and 10ml Binding Buffer amount suspension thalline is then added by every gram of wet thallus,
Under condition of ice bath in ultrasonication thalline (40W, work 2s rest 4s, crushes 40min), then 4 DEG C of 12000rpm centrifugations, collection
Clearly.By supernatant with after 0.22um membrane filtration, be added to through Binding Buffer it is equilibrated in Ni-NTA gravity posts,
4 DEG C are incubated after 10min, allow liquid to flow through filler through gravity, are removed successively with the Binding Buffer of the imidazoles of 20mM containing 10mM
Foreign protein, elutes destination protein, and collect destination protein with the Binding Buffer of the imidazoles containing 250mM.Collect 30ml or so
Destination protein after, centrifuge desalinations with 10KD 4 DEG C of 3500g of super filter tube.
3. the detection of destination protein
By SDS-PAGE method testing goal albumen, AaNGT Protein Detection results are shown in Fig. 2.Destination protein is N sugar
Based transferase AaNGT, its amino acid sequence is as shown in SEQ ID NO.1.
Embodiment 2：Applications of the N glycosyl transferases AaNGT in polypeptide is glycosylation modified
1. the measure of enzyme activity：
The peptide substrate for determining enzyme activity is the hexapeptide DANYTK with fluorescence labeling that Nanjing Jin Sirui companies synthesize, NGT's
Under catalysis, reacted respectively at UDP-Glc or UDP-Gal, reaction solution is detected through HPLC fluorescence detectors, reaction system such as table 1-3：
Table 1-3 AaNGT Enzyme activity assay reaction systems
Reaction result figure is shown in Fig. 3, it is found that AaNGT can be using UDP-Glc and UDP-Gal to the polypeptide containing glycosylation sequences
Modified, simple analysis is then carried out to product, mass spectral results figure is shown in Fig. 4.
2. the measure of optimal pH：
In order to probe into AaNGT optimal pH, we choose different pH buffer solution, HAc (5.0,6.0) PBS (6.0,7.0,
8.0) Tris (8.0,9.0,10.0) Hepes (7.0,8.0).And do 10 μ L reaction system, boil 5min after reaction 15min and stop
Only react, then analyzed with HPLC.Reaction system such as table 1-4：
The measure of table 1-4 optimal pHs
Reaction result figure is shown in Fig. 5, it is found that AaNGT activity in pH8.0 PBS is most strong.
3. the measure of optimum temperature：
In order to probe into AaNGT optimum temperatures, we do 10 μ L reaction system, and 15min is reacted at different temperature and (is divided
Not at 10 DEG C, reacted at 20 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 50 DEG C and 60 DEG C), then boil 5min and stop reaction, be used in combination
HPLC is analyzed, reaction system such as table 1-5：
The measure of table 1-5 optimum temperatures
Reaction result is shown in Fig. 6, it is found that AaNGT activity at 30 DEG C is highest.
4. influence of the metal ion to activity：
Metal ion in have chosen 8, influence of the research metal ion to AaNGT enzyme activity, reaction system such as table 1-6：
Influence of the table 1-6 metal ions to enzyme activity
Reaction result is shown in Fig. 7, it has been found that AaNGT is active in the case where there is non-metallic ion, wherein Cu2+With
Mn2+Ion has obvious inhibitory action for AaNGT activity.
5. glycosylate the comparison of efficiency：
In order to compare the difference of the glycosylation efficiency between AaNGT and ApNGT, following reaction system is designed, it is ensured that system
In various conditions it is all identical, reaction system such as table 1-7：
Table 1-7 glycosylates the comparison of efficiency
Reaction result is shown in Fig. 8, and we can substantially have found that the product that AaNGT catalysis is produced substantially will from HPLC result
Higher than the ApNGT reported for work, illustrate that AaNGT is higher than ApNGT using UDP-Glc ability.
6. glycosyl donor substrate specificity is analyzed：
5 kinds of UDP-Sugar derivatives are chosen, AaNGT glycosyl donor substrate specificity is studied, wherein UDP-Sugar spreads out
Biology is shown in Table 1-8, reaction system such as table 1-9：
Table 1-8 UDP-Sugar derivatives
Table 1-9 glycosyl donors substrate specificity is analyzed
Reaction HPLC results are shown in Fig. 9, it has been found that AaNGT can utilize UDP-GlcNH2, mass spectrum then is carried out to product
Checking, mass spectral results figure is shown in Figure 10, and mass spectral results prove that AaNGT utilizes UDP-GlcNH2。
Sequence table
<110>Shandong University
<120>A kind of N glycosyl transferases AaNGT and its application
<141>2017-6-20
<160>1
<210>1
<211> 621
<212>PRT
<213>Artificial sequence
<221>AaNGT amino acid sequence
<222>（1）…（621）
<400>1
MSRKKNPSVI QFEKAITEKN YEAACTELLD ILNKIDTNFG DIEGIDFDYP QQLETLMQDR 60
IVYFCTRMSN AITQLFCDPQ FSLSESGANR FFVVQRWLNL IFASSPYINA DHILQTYNCN 120
PERDSIYDIY LEPNKNVLMK FAVLYLPESN VNLNLDTMWE TDKNICGSLC FALQSPRFIG 180
TPAAFSKRST ILQWFPAKLE QFHVLDDLPS NISHDVYMHC SYDTAENKHN VKKALNQVIR 240
SHLLKCGWQD RQITQIGMRN GKPVMVVVLE HFHSSHSIYR THSTSMIAAR EQFYLIGLGN 300
NAVDQAGRDV FDEFHEFDGS NILKKLAFLK EMCEKNDAAV LYMPSIGMDL ATIFVSNARF 360
APIQVIALGH PATTHSEFIE YVIVEDDYVG SESCFSETLL RLPKDALPYV PSSLAPTDVQ 420
YVLRETPEVV NIGIAATTMK LNPYFLETLK TIRDRAKVKV HFHFALGQSI GITHPYVARF 480
IRSYLGDDAT AHPHSPYNRY LDILHNCDMM LNPFPFGNTN GIIDMVTLGL VGVCKTGPEV 540
HEHIDEGLFK RLGLPEWLIA DSVEDYIERA IRLAENHQER LALRRHIIEN NGLKTLFSGD 600
PSPMGKTLFA KLTEWRQTNG I 621
Claims (3)
1. a kind of N glycosyl transferases AaNGT, it is characterised in that：The N glycosyl transferases AaNGT derives from Haemophilus aphrophilus
(Aggregatibacter aphrophilus), its amino acid sequence is as shown in SEQ ID NO.1.
2. N glycosyl transferases AaNGT described in claim 1 is in the application to polypeptide progress N connections in glycosylation modified.
3. application as claimed in claim 2, it is characterised in that：Using AaNGT as a kind of glycosylation modified toolenzyme of polypeptide,
UDP-Glc or UDP-Gal are transferred to the polypeptide of the sequence containing N-X-S/T, glycopeptide is formed；Or AaNGT is by special glycosyl donor
UDP-GlcNH2It is transferred on the polypeptide containing N glycosylation sites, recycles other glycosyl transferases by GlcNH2Change into
GlcNAc, forms natural N sugar connections.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249353A (en) * | 2021-05-14 | 2021-08-13 | 山东大学 | N-glycosyltransferase mutant F13 and application thereof |
| CN113249352A (en) * | 2021-05-14 | 2021-08-13 | 山东大学 | N-glycosyltransferase mutant P1 and application thereof |
| CN116218806A (en) * | 2022-10-21 | 2023-06-06 | 山东大学 | N-glycosyltransferase mutant AaFQ and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013020988A1 (en) * | 2011-08-08 | 2013-02-14 | Eth Zurich | Pasteurellaceae vaccines |
| CN105505959A (en) * | 2016-01-11 | 2016-04-20 | 南开大学 | ApNGT gene of actinobacillus pleuropneumoniae and application of ApNGT gene |
-
2017
- 2017-06-26 CN CN201710497269.9A patent/CN107034202A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013020988A1 (en) * | 2011-08-08 | 2013-02-14 | Eth Zurich | Pasteurellaceae vaccines |
| CN105505959A (en) * | 2016-01-11 | 2016-04-20 | 南开大学 | ApNGT gene of actinobacillus pleuropneumoniae and application of ApNGT gene |
Non-Patent Citations (2)
| Title |
|---|
| REMPE KA ET AL: "Hmw1C［Aggregatibacter aphrophilus］，Rempe KA et al，GenBank:ALC78880.1", 《GENBANK》 * |
| REMPE KA ET AL: "Unconventional N-linked glycosylation promotes trimeric autotransporter function in Kingella kingae and Aggregatibacter aphrophilus", 《MBIO》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249353A (en) * | 2021-05-14 | 2021-08-13 | 山东大学 | N-glycosyltransferase mutant F13 and application thereof |
| CN113249352A (en) * | 2021-05-14 | 2021-08-13 | 山东大学 | N-glycosyltransferase mutant P1 and application thereof |
| CN113249352B (en) * | 2021-05-14 | 2022-03-22 | 山东大学 | N-glycosyltransferase mutant P1 and application thereof |
| CN113249353B (en) * | 2021-05-14 | 2022-03-22 | 山东大学 | N-glycosyltransferase mutant F13 and application thereof |
| CN116218806A (en) * | 2022-10-21 | 2023-06-06 | 山东大学 | N-glycosyltransferase mutant AaFQ and application thereof |
| CN116218806B (en) * | 2022-10-21 | 2024-05-10 | 山东大学 | N-glycosyltransferase mutant AaFQ and application thereof |
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