CN106191015A - The staphylococcus aureus sorting efficient mutant of enzyme A - Google Patents

The staphylococcus aureus sorting efficient mutant of enzyme A Download PDF

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CN106191015A
CN106191015A CN201610726374.0A CN201610726374A CN106191015A CN 106191015 A CN106191015 A CN 106191015A CN 201610726374 A CN201610726374 A CN 201610726374A CN 106191015 A CN106191015 A CN 106191015A
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mutant
enzyme
sorting enzyme
staphylococcus aureus
sorting
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CN106191015B (en
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陈鹏
陈龙
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Peking University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/2207—Sortase A (3.4.22.70)

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Abstract

The invention discloses the staphylococcus aureus sorting efficient mutant of enzyme A.By staphylococcus aureus sorting enzyme A gene and a kind of known mutations body gene, mutated library is built by fallibility PCR and site saturation mutation, utilize Screening Platform based on FRET (fluorescence resonance energy transfer), the sorting enzyme A mutant obtaining a series of catalysis activity raisings is integrated in screening and sudden change through too much taking turns, and these mutants comprise D124G, Y187L, E189R and/or F200L sudden change.These sorting enzyme A mutants both can promote the efficiency of reaction between catalysis LPXTG and atypia substrate α Gly, can promote the most again catalysis LPXTG and typical substrate α GlynBetween reaction efficiency.

Description

The staphylococcus aureus sorting efficient mutant of enzyme A
Technical field
The present invention relates to the protease orthogenesis of bioengineering field, particularly to flat by building high-throughout screening Platform, evolve obtained staphylococcus aureus sorting enzyme A mutant, its can be catalyzed efficiently LPXTG Yu α-Gly or α- GlynBetween coupled reaction.
Background technology
Staphylococcus aureus sorting enzyme A (Sa-SrtA) is the class cysteine generally existed in gram positive bacteria Transpeptidase, typically, it can be with catalytic substrate LPXTG (X represents arbitrary amino acid) and α-GlynBetween (oligomerization glycine) Coupled reaction, this reaction therefore be named as sort enzyme mediation coupled reaction (Sortase-Mediated Ligation, SML).Meanwhile, sorting enzyme A can also be catalyzed the reaction between LPXTG and α-Gly, but, it is catalyzed α-Gly relative to itnAnti- Should, reactivity is lower.
Utilize the coupled reaction of sorting enzyme mediation, it is possible to achieve the fixed point labelling of various protein and coupling, and then application Basic research and pharmaceutical industry in life sciences.But, owing to wild type sorting enzyme A catalytic efficiency is relatively low, based on high pass The orthogenesis of amount screening and the method for design and rational promote the activity of sorting enzyme A becomes a kind of necessary.In the research of forefathers Have also discovered 5 point mutation P94R/D160N/D165A/K190E/K196T (Chen I.et al.ProcNatl AcadSci USA 2011,108,11399-11404), its activity can be promoted to a certain extent.
Wild-type S. aureus bacterium sorting enzyme A is the protease comprising 206 amino acid residues, is forefathers Research in find, cut out 25 amino acid residues of nitrogen end truncate can in escherichia coli high level, the table of solubility Reach (Ton-That H.et al.ProcNatl AcadSci USA 1999,96,12,424 12429), divide the most under study for action Selecting enzyme A to be substantially all presented in this truncate, what the efficient mutant that the present invention relates to described is also this truncate The mutant of body.
Summary of the invention
The technical purpose of the present invention is to provide catalysis LPXTG with the α higher staphylococcus aureus of-Gly reactivity and divides Selecting enzyme A mutant, these mutants can also promote LPXTG Yu α-Gly further simultaneouslynBetween reaction activity.The present invention Construct the Screening Platform of FRET (fluorescence resonance energy transfer) based on two fluorescins, sort with wild-type S. aureus bacterium Enzyme A and P94R/D160N/D165A/K190E/K196T mutant thereof, respectively as the sorting enzyme 1 and 2 that sets out, are determined respectively To evolving and design and rational, final structure obtains active higher sorting enzyme A mutant so that it is can preferably be catalyzed LPXTG With α-Gly or α-GlynBetween coupled reaction.
The sorting enzyme A mutant that the present invention obtains both can promote known sorting enzyme A catalysis LPXTG and atypia substrate α- The efficiency of reaction between Gly, can promote again its catalysis LPXTG and typical substrate α-Gly simultaneouslynBetween reaction efficiency.
The raising of the sorting enzyme A mutant activity that the present invention is obtained, with kinetic constant K measuredcat, KmLPETGWith KmGGGOr KmGAGCharacterize, wherein, KcatRepresent the speed of reaction, KmLPETG、KmGGGAnd KmGAGRepresent enzyme respectively to substrate The recognition efficiency of LPETG, GGG and GAG.
Concrete, sorting enzyme A mutant provided by the present invention comprise one or more in following sudden change: D124G, Y187L, E189R and F200L.
The present invention provide sorting enzyme A mutant can also comprise one or more in following sudden change: P94R, D160N, D165A, K190E and K196T.
Currently preferred sorting enzyme A mutant is one of following mutant:
Mutant 1:P94R/D124G/D160N/D165A/K190E/K196T
Mutant 2:P94R/D160N/D165A/Y187L/E189R/K190E/K196T
Mutant 3:P94R/D124G/D160N/D165A/Y187L/E189R/K190E/K196T
Mutant 4:D124G/Y187L/E189R/F200L
Mutant 5:P94R/D124G/D160N/D165A/Y187L/E189R/K190E/K196T/F200L
Table 1 below gives wild type sorting enzyme A known mutations body P94R/D160N/D165A/K190E/K196T (to go out Send out sorting enzyme 2) and kinetic constant K of coupled reaction of mutant 4 and 5 catalysis LPXTG Yu α-Glycat, KmLPETGAnd KmGAG； Table 2 gives wild type sorting enzyme A, set out sorting enzyme 2 and above-mentioned five kinds of mutants catalysis LPXTG Yu α-GlynCoupled reaction Kinetic constant Kcat, KmLPETGAnd KmGGG, with Kcat/KmLPETGInstruction catalytic efficiency, it can be seen that the efficiency of each mutant All have greatly improved.
Table 1
Table 2
The method that the present invention is combined by orthogenesis and design and rational builds the sorting enzyme A obtaining being catalyzed activity raising Mutant, by wild-type S. aureus bacterium sorting enzyme A gene (GenBank accession number: BA000018, ORFID: SA2316) and the gene of a kind of known mutations body (P94R/D160N/D165A/K190E/K196T) sets out, use fallibility PCR and Site saturation mutation obtains sorting enzyme A mutated library, the Screening Platform based on FRET (fluorescence resonance energy transfer) that recycling builds, warp Too much the screening of wheel and the integration of sudden change obtain the sorting enzyme A mutant of a series of evolution, in the aminoacid sequence of sudden change Contain the one or more sudden changes in D124G, Y187L, E189R, F200L and known mutations.
Sorting enzyme A and mutant thereof that the embodiment of the present invention is expressed in prokaryotic expression system are that it clips 25 ammonia of nitrogen end The truncate form of base acid residue, wherein: include the aminoacid sequence such as sequence of the sorting enzyme A mutant 1 in 6 mutational sites In table shown in SEQ ID NO.1；Include in the aminoacid sequence such as sequence table of sorting enzyme A mutant 2 in 7 mutational sites Shown in SEQ ID NO.2；SEQ ID in the aminoacid sequence such as sequence table of the sorting enzyme A mutant 3 including 8 mutational sites Shown in NO.3；SEQ ID NO.4 institute in the aminoacid sequence such as sequence table of the sorting enzyme A mutant 4 including 4 mutational sites Show；Include the aminoacid sequence of sorting enzyme A mutant 5 in 9 mutational sites as shown in SEQ ID NO.5 in sequence table.Sequence The most each self-contained 181 amino acid residues of SEQ ID NO.1 to SEQ ID NO.5 in list, corresponding to wild type Staphylococcus Portugal 26th to the 206 amino acids residue sequence of grape coccus sorting enzyme A, said mutation site is also golden yellow relative to total length wild type For color staphylococcus sorting enzyme A.
The gene order that in sequence table, aminoacid sequence shown in SEQ ID NO.1 to SEQ ID NO.5 is corresponding is the most permissible Respectively as shown in SEQ ID NO.6 to SEQ ID NO.10, certainly, according to the degeneracy of codon, it is also possible to be utilize same Amino acid whose different codon and other nucleotide sequences of coding same protein of obtaining.
Carrier, cell and Host Strains containing these gene orders are the most within the scope of the present invention.
The present invention utilizes the Screening Platform of structure, golden yellow to built by the method for fallibility PCR and site saturation mutation The gene mutation library of color staphylococcus sorting enzyme A has carried out high flux screening, and obtained that catalytic efficiency improves a lot is prominent Variant, its catalysis LPXTG and typical substrate α-GlynAnd the efficiency of atypia substrate α-Gly has had the biggest lifting.
Accompanying drawing explanation
Fig. 1 shows the structure principle of sorting enzyme A Screening Platform based on FRET (fluorescence resonance energy transfer) principle.
Fig. 2 shows the effect of Screening Platform instruction sorting enzyme A activity.
Fig. 3 shows the site mutation impact for sorting enzyme A activity.
Fig. 4 shows wild type and kinetic constant K of mutant sorting enzyme A catalysis LPXTG Yu α-Gly reactioncat, KmLPETGWith KmGAG。
Fig. 5 shows that wild type and mutant sorting enzyme A are catalyzed LPXTG Yu α-GlynKinetic constant K of reactioncatWith KmLPETG。
Fig. 6 shows that wild type and mutant sorting enzyme A are catalyzed LPXTG Yu α-GlynKinetic constant K of reactionmGGG。
Detailed description of the invention
The culture medium prescription related in embodiment is as follows:
LB fluid medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH7.4.
Embodiment 1, utilize fallibility PCR build staphylococcus aureus sorting enzyme A libraries of random mutants
Utilizing fallibility PCR to introduce coding mutation to staphylococcus aureus sorting enzyme A gene in vitro, fallibility PCR has Concrete conditions in the establishment of a specific crime is as follows:
Pcr template is the plasmid comprising wild-type S. aureus bacterium sorting enzyme A gene, and this plasmid is by by wild type The gene (Ton-That H.et al.ProcNatl AcadSci USA 1999,96,12,424 12429) of sorting enzyme A sets out, Utilize sub-clone the 26th to the nucleotide sequence that the aminoacid sequence of 206 is corresponding is cloned into pet28a carrier NdeI and Obtain between XhoI restriction enzyme site.
Forward primer: 5 '-GGAATTCCATATGAAACCACATATCGATAATTATC-3 ' (SEQ ID NO.11)
Downstream primer: 5 '-GGTAGGCACTCGAGTTATTTGACTTCTGTAGCTAC-3 ' (SEQ ID NO.12)
PCR amplification condition: 95 DEG C of 5min；95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃10min.
Fallibility pcr amplification product is through PCR primer Purification Kit, and restricted enzyme NdeI and XhoI is the most right PCR primer and pet28a carrier carry out enzyme action, connect, and convert to escherichia coli DH10B, coat containing 100 μ g/mL cards that is mould The LB flat board of element, after 37 DEG C of incubators grow 12 hours, collects all clones, is diluted to continued growth in LB liquid medium, Extract plasmid after 12 hours, obtain mutated library.By the Plastid transformation that obtains to e. coli bl21 (DE3), 37 DEG C of incubators After growing 12 hours, obtain the expression bacterium of mutated library, stand-by.
Embodiment 2, point mutation PCR build staphylococcus aureus sorting saturation mutation library, enzyme A site
Utilize the method for point mutation PCR to the two of the P94R/D160N/D165A/K190E/K196T mutant of sorting enzyme A Individual site Tyr187 and Glu189 introduces saturation mutation, and PCR condition is as follows:
Pcr template is the gene setting out and sorting enzyme 2, by sorting enzyme A gene containing wild type in embodiment 1 Obtained by pet28a plasmid encoding mutant.
Forward primer: 5 '-CATTAATTACTTGTGATGATNNKAATNNKGAGACAGGCGTTTGGG-3 ' (SEQ ID NO.13)
Downstream primer: 5 '-CCCAAACGCCTGTCTCMNNATTMNNATCATCACAAGTAATTAATG-3 ' (SEQ ID NO.14)
In above-mentioned primer, N represents the one in tetra-kinds of bases of A, C, G, T, and K represents G or T, M and represents A or C.
PCR amplification condition: 95 DEG C of 5min；95 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 7min, 23 circulations；72℃10min.
Point mutation PCR primer, through DpnI enzyme action, converts in escherichia coli DMT bacterial strain, coats containing 100 μ g/mL cards The LB flat board of that mycin, after 37 DEG C of incubators grow 12 hours, collects all clones, is diluted in LB liquid medium continue life Long, extract plasmid after 12 hours, obtain the saturation mutation library of dibit point.By the Plastid transformation that obtains to e. coli bl21 (DE3), after 37 DEG C of incubators grow 12 hours, the expression bacterium in saturation mutation library is obtained, stand-by.
Embodiment 3, screening system based on FRET (fluorescence resonance energy transfer) build and checking
Utilize the method fluorescence egg of amalgamation and expression eGFP-LPETG and G-cpV on pet28a carrier respectively of sub-clone In vain, utilizing 6 × His label to be purified, the albumen that purification obtains carries out sorting the coupled reaction of enzyme mediation, as it is shown in figure 1, EGFP and cpV albumen is suitable FRET (fluorescence resonance energy transfer) (FRET) donor and receptor, when sorting enzyme A mediate two albumen it Between coupled reaction time, due to furthering of two albumen distances, the transferring efficiency of fluorescence resonance energy between two albumen promotes, Sorting enzymatic activity is the highest, then fluorescent functional energy transfer efficiency is the highest.Utilize wild type sorting enzyme A and the sorting enzyme 2 that sets out to sieve Platform is selected to verify, it was demonstrated that sorting enzyme A activity is directly proportional to transferring efficiency of fluorescence resonance energy, as shown in Figure 2.
The screening of embodiment 4, high catalytic activity sorting enzyme A mutant
The gene library that picking embodiment 1 and 2 builds, containing 1ml LB (containing 100 μ g/ml kanamycin) culture medium In deep hole 96 orifice plate, incubated overnight in 37 DEG C of shaking tables, 1:100 is transferred in new deep hole 96 orifice plate, and 37 DEG C of shaking tables are cultivated extremely OD600Reaching about 0.6, add 1mM IPTG induction, 37 DEG C are continued to cultivate 3 hours.4700rmp is centrifuged 10 minutes, abandons supernatant, often Hole adds 200 μ L lysates (30mM Tris, 150mM NaCl, 5mM CaCl2, 1%Triton X-100,0.15mg/mL are molten Bacterium enzyme), 37 DEG C of shaking tables crack 2 hours, and centrifugal segregation cell debris obtains cell pyrolysis liquid.200 μ L reaction systems add 50 μ L cell pyrolysis liquid, 100 μMs of eGFP-LPETG-His6,200 μMs of G-cpV, 37 DEG C are reacted 2 hours.Utilize microplate reader, 435nm Excite, collect 475nm and 525nm fluorescence, represent different fluorescence resonance energies by 525nm and 475nm fluorescence intensity ratio Transfer efficiency, thus obtain the mutant of different catalytic efficiency.As it is shown on figure 3, the present invention has obtained four can promote sorting The sudden change of enzyme A activity, comprises D124G, Y187L, E189R and F200L.By these four suddenly change in one or more sudden changes with go out Five sudden changes sending out sorting enzyme 2 are integrated, and obtain final mutant.
Embodiment 5, staphylococcus aureus sorting enzyme A determination of activity
Staphylococcus aureus sorting enzyme A determination of activity is according to method (the Kruger R.G. of document；Dostal P； McCafferty D.G.Anal Biochem 2004,326:42 48), except for the difference that, measure LPXTG Yu α-GlynReaction Kinetic constant, using GGG as reaction substrate；Measure the kinetic constant of LPXTG Yu α-Gly reaction, using GAG as the reaction end Thing.As shown in Figure 4, with Abz-LPETGK (Dnp)-CONH2It is measured as substrate with GAG, mutant sorting enzyme A catalysis Activity (the K of LPETG Yu α-Gly reactioncat/KmLPETG) obtain about 7～79 times of different liftings relative to wild type, best Mutant also has the lifting more than 5 times relative to the sorting enzyme 2 that sets out, and it is for the identification (K of GAG simultaneouslymGGG) also there are 1.3 times of left sides Right lifting.As it can be seen in figures 5 and 6, with Abz-LPETGK (Dnp)-CONH2It is measured as substrate with GGG, it can be seen that phase Enzyme A, mutant sorting enzyme A are sorted for wild type and is catalyzed LPETG Yu α-GlynActivity (the K of reactioncat/KmLPETG) relative to open country Raw type has obtained 20～245 times of different liftings, and best mutant also has the lifting of nearly 11 times relative to the sorting enzyme 2 that sets out, It is for the identification (K of GGG simultaneouslymGGG) also there is the lifting of about 2 times.

Claims (9)

1. staphylococcus aureus sorting enzyme A a mutant, comprise one or more in following sudden change: D124G, Y187L, E189R and F200L.
2. the mutant of staphylococcus aureus sorting enzyme A as claimed in claim 1, it is characterised in that described mutant is also Comprise one or more in following sudden change: P94R, D160N, D165A, K190E and K196T.
3. the mutant of staphylococcus aureus sorting enzyme A as claimed in claim 1, it is characterised in that described mutant is One of following mutant:
Mutant 1:P94R/D124G/D160N/D165A/K190E/K196T；
Mutant 2:P94R/D160N/D165A/Y187L/E189R/K190E/K196T；
Mutant 3:P94R/D124G/D160N/D165A/Y187L/E189R/K190E/K196T；
Mutant 4:D124G/Y187L/E189R/F200L；
Mutant 5:P94R/D124G/D160N/D165A/Y187L/E189R/K190E/K196T/F200L.
4. the mutant of staphylococcus aureus sorting enzyme A as claimed in claim 1, it is characterised in that described mutant is Comprise in sequence table the protein of aminoacid sequence shown in any one in SEQ ID NO.1 to SEQ ID NO.5.
5. the gene of the mutant of staphylococcus aureus sorting enzyme A described in Claims 1 to 4 any one.
6. gene as claimed in claim 5, it is characterised in that described gene has SEQ ID NO.6 to SEQ in sequence table The nucleotide sequence that in ID NO.10, any one is shown.
7. comprise the carrier of gene described in claim 5, cell or Host Strains.
8. described in Claims 1 to 4 any one staphylococcus aureus sorting enzyme A mutant catalytic substrate LPXTG with The application in coupled reaction between α-Gly, wherein X represents arbitrary amino acid.
9. described in Claims 1 to 4 any one staphylococcus aureus sorting enzyme A mutant catalytic substrate LPXTG with Oligomerization glycine α-GlynBetween coupled reaction in application, wherein X represents arbitrary amino acid.
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CN109797194A (en) * 2019-01-24 2019-05-24 北京大学 It marks cell membrane surface and studies the enzyme and method of cell-ECM interaction

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Publication number Priority date Publication date Assignee Title
CN106636019A (en) * 2016-12-30 2017-05-10 江南大学 Synthesis of (S)-1-Phenyl-1,2-ethanediol through efficient catalysis of sortase A mediated (S)-carbonyl reductase oligomer
CN106636019B (en) * 2016-12-30 2019-12-10 江南大学 High-efficiency catalytic synthesis of (S) -phenyl glycol by using (S) -carbonyl reductase oligomer mediated by Sortase A
CN109797194A (en) * 2019-01-24 2019-05-24 北京大学 It marks cell membrane surface and studies the enzyme and method of cell-ECM interaction

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Application publication date: 20161207

Assignee: WEST LAKE BIOMEDICAL TECHNOLOGY (HANGZHOU) Co.,Ltd.

Assignor: Peking University

Contract record no.: X2022980023323

Denomination of invention: Highly efficient mutant of Staphylococcus aureus sorting enzyme A

Granted publication date: 20190913

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Record date: 20221208

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