CN106191015B - The efficient mutant of staphylococcus aureus sorting enzyme A - Google Patents

The efficient mutant of staphylococcus aureus sorting enzyme A Download PDF

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CN106191015B
CN106191015B CN201610726374.0A CN201610726374A CN106191015B CN 106191015 B CN106191015 B CN 106191015B CN 201610726374 A CN201610726374 A CN 201610726374A CN 106191015 B CN106191015 B CN 106191015B
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mutant
sorting enzyme
seq
staphylococcus aureus
enzyme
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CN106191015A (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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Abstract

The invention discloses the efficient mutant of staphylococcus aureus sorting enzyme A.By staphylococcus aureus sorting enzyme A gene and a kind of known mutations body gene, mutated library is constructed by fallibility PCR and site saturation mutation, utilize the Screening Platform based on fluorescence resonance energy transfer, screening and mutation integration through excessively taking turns obtain a series of sorting enzyme A mutant that catalytic activity improve, these mutant are mutated comprising D124G, Y187L, E189R and/or F200L.These sorting enzymes A mutant can not only promote the efficiency reacted between catalysis LPXTG and atypia substrate α-Gly, but also can promote catalysis LPXTG and typical substrate α-GlynBetween the efficiency reacted.

Description

The efficient mutant of staphylococcus aureus sorting enzyme A
Technical field
The present invention relates to the albumen directed enzyme evolutions of bioengineering field, in particular to flat by the high-throughput screening of building Platform, evolution have obtained the mutant of staphylococcus aureus sorting enzyme A, can efficiently be catalyzed LPXTG and α-Gly or α- GlynBetween connection reaction.
Background technique
Staphylococcus aureus sorting enzyme A (Sa-SrtA) is a kind of cysteine generally existing in gram-positive bacteria Transpeptidase typically can be with catalysis substrate LPXTG (X represents arbitrary amino acid) and α-GlynBetween (oligomerization glycine) Connection reaction, this reaction therefore be named as sorting enzyme mediation connection reaction (Sortase-Mediated Ligation, SML).Meanwhile sorting enzyme A can also be catalyzed reacting between LPXTG and α-Gly, however, being catalyzed α-Gly relative to itnIt is anti- It answers, reactivity is lower.
It is reacted using the connection that sorting enzyme mediates, fixed point label and the coupling of various protein may be implemented, and then applied Basic research and pharmaceutical industry in life science.However, being based on high pass since wild type sorting enzyme A catalytic efficiency is lower The activity that the method for the directed evolution and design and rational of measuring screening promotes sorting enzyme A becomes a kind of necessity.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 in forefathers Research in find, cut out 25 amino acid residues of nitrogen end truncate can in Escherichia coli high level, soluble table Up to (Ton-That H.et al.ProcNatl AcadSci USA 1999,96,12424-12429), therefore divide under study for action It selects enzyme A to be substantially all to exist in the form of this truncate, efficient mutant description of the present invention is also this truncation The mutant of body.
Summary of the invention
Technical purpose of the invention is to provide catalysis LPXTG and the higher staphylococcus aureus of α-Gly reactivity point Enzyme A mutant is selected, while these mutant can also further promote LPXTG and α-GlynBetween the activity reacted.The present invention The Screening Platform of the fluorescence resonance energy transfer based on two fluorescins is constructed, with the sorting of wild-type S. aureus bacterium Enzyme A and its P94R/D160N/D165A/K190E/K196T mutant are determined respectively respectively as sorting enzyme 1 and 2 of setting out To evolution and design and rational, final building obtains the higher sorting enzyme A mutant of activity, allows to preferably be catalyzed LPXTG With α-Gly or α-GlynBetween connection reaction.
The sorting enzyme A mutant that the present invention obtains can both promote known sorting enzyme A catalysis LPXTG and atypia substrate α- The efficiency reacted between Gly, while it can be promoted again and be catalyzed LPXTG and typical substrate α-GlynBetween the efficiency reacted.
The raising of present invention sorting enzyme A mutant activity obtained, with the kinetic constant K of measurementcat, KmLPETGWith KmGGGOr KmGAGIt is characterized, wherein KcatRepresent the rate of reaction, KmLPETG、KmGGGAnd KmGAGEnzyme is respectively represented to substrate The recognition efficiency of LPETG, GGG and GAG.
Specifically, sorting enzyme A mutant provided by the present invention includes one of following mutation or a variety of: D124G, Y187L, E189R and F200L.
Sorting enzyme A mutant provided by the invention can also include one of following mutation or a variety of: 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 (out Hair sorting enzyme 2) and mutant 4 and 5 be catalyzed the kinetic constant K that reacts with the connection of α-Gly of LPXTGcat, KmLPETGAnd KmGAG； Table 2 gives wild type sorting enzyme A, sorting enzyme of setting out 2 and above-mentioned five kinds of mutant catalysis LPXTG and α-GlynConnection reaction Kinetic constant Kcat, KmLPETGAnd KmGGG, with Kcat/KmLPETGIndicate catalytic efficiency, it can be seen that the efficiency of each mutant All have greatly improved.
Table 1
Table 2
The present invention constructs to obtain the sorting enzyme A of catalytic activity raising by the method that directed evolution and design and rational combine Mutant, by wild-type S. aureus bacterium sorting enzyme A gene (GenBank accession number: BA000018, ORFID: SA2316) and a kind of gene of known mutations body (P94R/D160N/D165A/K190E/K196T) sets out, with fallibility PCR and Site saturation mutation obtains sorting enzyme A mutated library, recycles the Screening Platform based on fluorescence resonance energy transfer of building, warp The integration of the screening and mutation excessively taken turns obtains a series of sorting enzyme A mutant of evolution, in the amino acid sequence of mutation D124G, Y187L, E189R are contained, one or more mutation in F200L and known mutations.
The sorting enzyme A and its mutant 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, in which: include the amino acid sequence such as sequence of the sorting enzyme A mutant 1 in 6 mutational sites In table shown in SEQ ID NO.1；It include in the amino acid sequence such as sequence table of the sorting enzyme A mutant 2 in 7 mutational sites Shown in SEQ ID NO.2；It include SEQ ID in the amino acid sequence such as sequence table of the sorting enzyme A mutant 3 in 8 mutational sites Shown in NO.3；It include the amino acid sequence such as SEQ ID NO.4 institute in sequence table of the sorting enzyme A mutant 4 in 4 mutational sites Show；It include the amino acid sequence of the sorting enzyme A mutant 5 in 9 mutational sites as shown in SEQ ID NO.5 in sequence table.Sequence SEQ ID NO.1 to SEQ ID NO.5 respectively contains 181 amino acid residues in list, corresponds to wild type Staphylococcus Portugal The 26th to the 206 amino acids residue sequence of grape coccus sorting enzyme A, above-mentioned mutational site are also golden yellow relative to overall length wild type For color staphylococcus sorting enzyme A.
The corresponding gene order of amino acid sequence shown in SEQ ID NO.1 to SEQ ID NO.5 successively can be in sequence table 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 using same The different codons of amino acid and other nucleotide sequences of coding same protein obtained.
Carrier, cell and host strain containing these gene orders are also within the scope of the present invention.
The present invention constructs the method by fallibility PCR and site saturation mutation golden yellow using the Screening Platform of building 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, catalysis LPXTG and typical substrate α-GlynAnd the efficiency of atypia substrate α-Gly has very big promotion.
Detailed description of the invention
Fig. 1 shows the building principle of the sorting enzyme A Screening Platform based on fluorescence resonance energy transfer principle.
Fig. 2 shows that Screening Platform indicates the active effect of sorting enzyme A.
Fig. 3 shows site mutation influence active for sorting enzyme A.
Fig. 4 shows wild type and the kinetic constant K that mutant sorting enzyme A catalysis LPXTG is reacted with α-Glycat, KmLPETGWith KmGAG。
Fig. 5 shows wild type and mutant sorting enzyme A catalysis LPXTG and α-GlynThe kinetic constant K of reactioncatWith KmLPETG。
Fig. 6 shows wild type and mutant sorting enzyme A catalysis LPXTG and α-GlynThe kinetic constant K of reactionmGGG。
Specific embodiment
Culture medium prescription involved in embodiment is as follows:
LB liquid medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH7.4.
Embodiment 1 constructs staphylococcus aureus sorting enzyme A libraries of random mutants using fallibility PCR
Coding mutation, fallibility PCR tool are introduced to staphylococcus aureus sorting enzyme A gene in vitro using fallibility PCR 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, this plasmid is by by wild type The gene (Ton-That H.et al.ProcNatl AcadSci USA 1999,96,12424-12429) of sorting enzyme A sets out, Using subclone by the 26th to the 206 corresponding nucleotide sequences of amino acid sequence be cloned into pet28a carrier NdeI and It is obtained between XhoI restriction enzyme site.
Upstream 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.
For fallibility pcr amplification product through PCR product Purification Kit, restriction enzyme NdeI and XhoI is right respectively PCR product and pet28a carrier carry out digestion, connection, conversion to Escherichia coli DH10B, are coated on that is mould containing 100 μ g/mL cards The LB plate of element collects whole clones, is diluted to continued growth in LB liquid medium after 37 DEG C of incubators are grown 12 hours, Plasmid is extracted after 12 hours, obtains mutated library.Obtained plasmid is converted to e. coli bl21 (DE3), 37 DEG C of incubators After growth 12 hours, the expression bacterium of mutated library is obtained, for use.
Embodiment 2, point mutation PCR construct the site staphylococcus aureus sorting enzyme A saturation mutation library
Using point mutation PCR P94R/D160N/D165A/K190E/K196T mutant from method to sorting enzyme A two A site Tyr187 and Glu189 introduces saturation mutation, and PCR condition is as follows:
Pcr template is the gene of sorting enzyme 2 of setting out, by will contain wild type sorting enzyme A gene in embodiment 1 Obtained by pet28a plasmid encoding mutant.
Upstream primer: 5 '-CATTAATTACTTGTGATGATNNKAATNNKGAGACAGGCGTTTGGG-3 ' (SEQ ID NO.13)
Downstream primer: 5 '-CCCAAACGCCTGTCTCMNNATTMNNATCATCACAAGTAATTAATG-3 ' (SEQ ID NO.14)
In above-mentioned primer, N represents one of tetra- kinds of bases of A, C, G, T, and K represents G or T, and M 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 product is converted in Escherichia coli DMT bacterial strain, is coated on containing 100 μ g/mL cards through DpnI digestion The LB plate of that mycin collects whole clones, is diluted to and continues to give birth in LB liquid medium after 37 DEG C of incubators are grown 12 hours It is long, plasmid is extracted after 12 hours, obtains the saturation mutation library of double site.Obtained plasmid is converted to e. coli bl21 (DE3), after 37 DEG C of incubators are grown 12 hours, the expression bacterium in saturation mutation library is obtained, for use.
Embodiment 3, screening system building and verifying based on fluorescence resonance energy transfer
Using subclone method respectively on pet28a carrier amalgamation and expression eGFP-LPETG and G-cpV fluorescence egg It is white, it is purified using 6 × His label, the albumen purified carries out the connection reaction of sorting enzyme mediation, as shown in Figure 1, EGFP and cpV albumen is suitable fluorescence resonance energy transfer (FRET) donor and receptor, when sorting enzyme A mediate two albumen it Between connection reaction when, due to furthering for two albumen distances, the transferring efficiency of fluorescence resonance energy between two albumen is promoted, Sorting enzymatic activity is higher, then fluorescent functional energy transfer efficiency is higher.Utilize wild type sorting enzyme A and 2 pairs of the sorting enzyme sieves that set out Platform is selected to be verified, 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 of picking Examples 1 and 2 building is containing 1ml LB (containing 100 μ g/ml kanamycins) culture medium It in 96 orifice plate of deep hole, is incubated overnight in 37 DEG C of shaking tables, 1:100 is transferred in new 96 orifice plate of deep hole, and 37 DEG C of shaking table cultures are extremely OD600Up to 0.6 or so, 1mM IPTG induction is added, 37 DEG C are continued culture 3 hours.4700rmp is centrifuged 10 minutes, abandons supernatant, often 200 μ L lysates (30mM Tris, 150mM NaCl, 5mM CaCl is added in hole2, 1%Triton X-100,0.15mg/mL be molten Bacterium enzyme), 37 DEG C of shaking tables crack 2 hours, and centrifugation removal cell fragment obtains cell pyrolysis liquid.50 are added in 200 μ L reaction systems μ L cell pyrolysis liquid, 100 μM of eGFP-LPETG-His6,200 μM of G-cpV, 37 DEG C are reacted 2 hours.Utilize microplate reader, 435nm Excitation, collects 475nm and 525nm fluorescence, represents different fluorescence resonance energies by 525nm and 475nm fluorescence intensity ratio Transfer efficiency, to obtain the mutant of different catalytic efficiencies.As shown in figure 3, the present invention, which has obtained four, can promote sorting The active mutation of enzyme A includes D124G, Y187L, E189R and F200L.By one or more mutation in this four mutation and go out Five mutation of hair sorting enzyme 2 are integrated, and final mutant is obtained.
Embodiment 5, staphylococcus aureus sorting enzyme A determination of activity
Method (Kruger R.G. of the staphylococcus aureus sorting enzyme A determination of activity according to document；Dostal P； McCafferty D.G.Anal Biochem 2004,326:42-48), unlike, measurement LPXTG and α-GlynReaction Kinetic constant, using GGG as reaction substrate；The kinetic constant that measurement LPXTG is reacted with α-Gly, using GAG as reaction bottom Object.As shown in figure 4, with Abz-LPETGK (Dnp)-CONH2It is measured with GAG as substrate, mutant sorting enzyme A catalysis Activity (the K that LPETG is reacted with α-Glycat/KmLPETG) relative to wild type about 7~79 times of different promotions are obtained, it is best Mutant also has a promotion greater than 5 times relative to sorting enzyme 2 of setting out, while it is for the identification (K of GAGmGGG) also there are 1.3 times of left sides Right promotion.As it can be seen in figures 5 and 6, with Abz-LPETGK (Dnp)-CONH2It is measured with GGG as substrate, it can be seen that phase LPETG and α-Gly is catalyzed for wild type sorting enzyme A, mutant sorting enzyme AnActivity (the K of reactioncat/KmLPETG) relative to open country Raw type has obtained 20~245 times of different promotions, and best mutant also has nearly 11 times of promotion relative to sorting enzyme 2 of setting out, Its identification (K for GGG simultaneouslymGGG) also there is 2 times or so of promotion.

Claims (7)

1. a kind of mutant of staphylococcus aureus sorting enzyme A, is one of following mutant:
Mutant D124G；
Mutant D124G, F200L；
Mutant 1:P94R, D124G, D160N, D165A, K190E and K196T；
Mutant 3:P94R, D124G, D160N, D165A, Y187L, E189R, K190E and K196T；
Mutant 4:D124G, Y187L, E189R and F200L；
Mutant 5:P94R, D124G, D160N, D165A, Y187L, E189R, K190E, K196T and F200L；
All mutational sites are for overall length wild-type S. aureus bacterium sorting enzyme A.
2. the mutant of staphylococcus aureus sorting enzyme A as described in claim 1, which is characterized in that the mutant is Sequence is SEQ ID NO.1 in sequence table, shown in any one in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 The protein of amino acid sequence.
3. the gene of the mutant of staphylococcus aureus sorting enzyme A as claimed in claim 1 or 2.
4. gene as claimed in claim 3, which is characterized in that the sequence of the gene be sequence table in SEQ ID NO.6, Nucleotide sequence shown in any one in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.
5. carrier, cell or host strain comprising gene described in claim 3.
6. the mutant of staphylococcus aureus sorting enzyme A as claimed in claim 1 or 2 is between catalysis substrate LPXTG and α-Gly Connection reaction in application, wherein X represents arbitrary amino acid.
7. the mutant of staphylococcus aureus sorting enzyme A as claimed in claim 1 or 2 is in catalysis substrate LPXTG and the sweet ammonia of oligomerization Sour α-GlynBetween connection reaction in application, wherein X represents arbitrary amino acid.
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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
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WO2021185360A1 (en) * 2020-03-20 2021-09-23 Westlake Therapeutics (Hangzhou) Co., Limited Novel truncated sortase variants

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