CN114149980A

CN114149980A - Novel protein biotin ligase and proximity labeling system PhastID based on same

Info

Publication number: CN114149980A
Application number: CN202111302493.0A
Authority: CN
Inventors: 松阳洲; 冯然; 刘峰; 吴苏; 李克成; 李影影; 周志芬; 李若菲; 刘熠
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2022-03-08
Anticipated expiration: 2041-11-04
Also published as: CN114149980B

Abstract

The invention discloses a novel protein biotin ligase and a proximity labeling system PhastID based on the same. Specifically disclosed are protein biotin ligase BPL, a mutant protein biotin ligase BPL after modification, and a proximal protein labeling system PhastID based on the biotin ligase BPL or BPL. Compared with the traditional BioID proximity labeling system, the labeling system containing the protein biotin ligase BPL has higher labeling strength, shorter required labeling time and larger application prospect.

Description

Novel protein biotin ligase and proximity labeling system PhastID based on same

Technical Field

The invention relates to the technical field of genetic engineering, in particular to novel protein biotin ligase BPL, mutant protein biotin ligase fusion protein BPL and a proximity labeling system PhastID based on the protein biotin ligase BPL or BPL.

Background

Protein Biotin ligase can label Biotin onto its substrate by activating Biotin (Biotin) to form Biotinyl-5 '-adenylate Biotinyl-5' -AMP. Through artificial modification, the ligase can lose specific labeling on a substrate, and the released Biotinyl-5' -AMP is extremely unstable in chemical property and can randomly react with adjacent lysine residues to covalently label biotin, wherein the range is within about 10 nm. And the high affinity and stability between biotin and streptavidin are combined, and the protein can be traced, purified and the like. The labeling radius is about 10nm, and the distance is just a precondition for Protein-Protein Interaction (PPI), so the method has wide application prospect in proteomics. In recent years, the development of a technology for labeling adjacent proteins by using Biotin ligase is rapid, the technology firstly forms fusion protein by using target protein and the Biotin ligase, after Biotin labeling is carried out for a certain time, the protein labeled by the Biotin ligase around the target protein is purified by using a Biotin-streptavidin system, and then the protein having interaction with the target protein is further identified by using a high-throughput method. Currently, there are several species of biotin ligases, which have been mutated to develop proximity labeling techniques, including BioID (BirA, from Escherichia coli, ref.: Roux K J, Kim D I, Raida M, et al.A. promoter biotin fusion proteins and proteins in mammalian cells [ J ]. J Cell Biol 2012,196(6): 801) BioID2(BPL, from Aquifex aeolicus, ref.: Kim D I, Jensen S C, Noble A, et al.a. animal promoter proteins for protein labeling [ J ]. Molecular biology, of Bacillus Cell proteins, et al.: BioID 2016, Ser. K8, Cell proteins for BioID proteins [ J ]. K8, M7, Ser. K7, M7, K, reference documents: branon T C, Bosch J A, Sanchez AD, et al, efficient promotion labeling in living cells and organisms with TurboID [ J ]. Nature biotechnology,2018,36(9): 880-. However, these proximity marking techniques have certain drawbacks, such as: the labeling capacity is weak, so the time required is long, and the capacity is weak when the instantaneous or weak interaction protein is detected; or the protein molecular weight of the enzyme is larger. Therefore, it is very important to find a new type of biological ligase which is more advantageous in structure and function.

Disclosure of Invention

The present invention aims to overcome the above defects and shortcomings of the prior art and provide a novel protein biotin ligase BPL.

The second objective of the invention is to provide a mutant protein biotin ligase BPL.

The third purpose of the invention is to provide the application of the protein biotin ligase BPL or mutant protein biotin ligase BPL.

The fourth object of the present invention is to provide a proximity labeling system comprising the protein biotin ligase BPL or the mutant protein biotin ligase BPL.

A fifth object of the invention is to provide an application of the proximity marking system.

The above object of the present invention is achieved by the following technical solutions:

a Protein Biotin Ligase (BPL), including BPL derived from the archaebacterium pyropoccus horikoshii, referred to as PhBPL; BPL from the archaebacterium pyropoccus kukukukuulkanii, referred to as PkBPL; or BPL from the archaebacterium methanodococcus fervens, referred to as MfBPL;

(a) the amino acid sequences are respectively and sequentially shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;

or

(b) A protein which has more than 70 percent of homology with the amino acid sequence of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 and has the activity of biotin protein ligase.

A mutant protein biotin ligase BPL which is a mutant of protein biotin ligase BPL (PhBPL, PkBPL, MfBPL); specifically, the mutation of the PhBPL R48 amino acid residue in SEQ ID NO. 1: the R48G or R48S mutation, namely PhBPL (R48G) or PhBPL (R48S), referred to as PhBPL; or the mutation of the amino acid residue of PkBPL R48 described in SEQ ID NO. 2: R48G or R48S mutations, i.e. PkBPL (R48G) or PkBPL (R48S), referred to as PkBPL; or MfBPL R40 amino acid residue according to SEQ ID NO. 3: R40G, MfBPL (R40G), is referred to as MfBPL.

The invention mutates PhBPL R48 amino acid residue, PkBPL R48 amino acid residue and MfBPL R40 amino acid residue, thus obtaining protein biotin ligase BPL which has higher labeling strength and shorter required labeling time compared with the traditional BioID.

The invention also provides a nucleotide sequence for coding the mutant protein biotin ligase BPL.

Preferably, the coding nucleotide sequences of PhBPL (R48G), PkBPL (R48G) and MfBPL (R40G) are shown in SEQ ID NO. 4-6 in sequence.

A recombinant expression vector comprising said mutein biotin ligase BPL encoding nucleotide sequence. For example: pcDNA3.1(-) -BPL, pLenti-HA-BPL.

A host cell comprising the recombinant expression vector. For example: 293T cells, HEK293T cells, Hela cells.

Each of the above-mentioned protein biotin ligase BPL can be individually established into a set of BPL labeling system. Specifically, BPL rapidly catalyzes the formation and release of biotin-5' -AMP from ATP and biotin, which reacts with and covalently binds biotin to lysine residues of adjacent proteins, thereby biotin-labeling adjacent proteins in vivo or in vitro in the presence of biotin.

The invention thus protects the use of the aforementioned protein biotin ligase BPL or mutant protein biotin ligase BPL for adjacent protein labeling or for the preparation of adjacent protein labeling systems.

A proximal protein labeling system comprising the protein biotin ligase BPL or the mutein biotin ligase BPL. In particular to a novel protein biotin ligase BPL which comprises a Bait protein (Bait protein) to be researched and is expressed at the N-end or the C-end of the Bait protein in a fusion mode.

A method for labeling adjacent protein includes such steps as forming fusion protein by target protein to be researched and the mutant protein biotin ligase BPL, labeling with biotin, purifying the protein labeled by biotin ligase around the target protein by biotin-streptavidin system, and high-throughput identification of the protein interacted with the target protein.

The method specifically comprises the following steps:

(1) the protein biotin ligase BPL is expressed by fusion at the N end or the C end of the 'bait protein' to be researched by a recombination method or a knock-in method and the like;

(2) in vitro or in vivo, under the condition that substrates such as ATP and Biotin exist, labeling for more than 15 min;

(3) lysing the cells or tissues and affinity purifying the Biotin-labeled protein with (Strept-) avidin coupled to a solid phase;

(4) the labeled protein is identified by ELISA, Western Blot, mass spectrometry and the like to find a new interacting protein.

Preferably, the biotin concentration is 5 μ M to 50 μ M.

Preferably, the marking time is 10min to 16 h.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a protein biotin ligase BPL, a modified mutant protein biotin ligase BPL and a proximity labeling system based on the protein biotin ligase BPL. The labeling system comprising protein biotin ligase BPL has higher labeling intensity and requires shorter labeling time than the conventional BioID proximity labeling system. Has wide application prospect.

Drawings

FIG. 1 is a diagram showing the detection results of Western blot (Western blot) of novel biotinylated protein (left is a detection result diagram, and right is a statistical diagram).

FIG. 2 is a graph (FIG. A) and a statistical graph (FIG. B) showing the results of Western blotting (Western blot) detection of novel biotinases at different times.

Fig. 3 is a graph comparing the labeling intensity of the novel biotinidase PhBPL with nuclear localization signal and the BioID2 (left is a graph of the detection results, right is a statistical graph).

FIG. 4 is a graph showing the detection results of the novel biotinidase in different concentrations in the labeled protein immunoblotting (Western blot).

FIG. 5 is a graph showing the in vitro detection of the interaction of the novel biotinidase fusion protein (TRF1) with the telomere binding protein RAP1 using streptavidin.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 production of the protein biotin ligase BPL (PhBPL, PkBPL and MfBPL)

Obtaining biotin ligase sequences of various species on NCBI, including BPL from archaea Pyrococcus horikoshii, called PhBPL, the amino acid sequence of which is shown in SEQ ID NO. 1; BPL derived from archaea Pyrococcus kukukukulukanii, called PkBPL, and the amino acid sequence of the BPL is shown as SEQ ID NO. 2; and BPL derived from archaea Methanocadococcus fervens, named MfBPL, the amino acid sequence of which is shown in SEQ ID NO. 3; analyzing the GNGR motif (N represents basic amino acid), and mutating the protein into a GNGG form; PhBPL R48 amino acid residue mutation: the R48G or R48S mutation, namely PhBPL (R48G) or PhBPL (R48S), referred to as PhBPL; PkBPL R48 amino acid residue mutation: R48G or R48S mutations, i.e. PkBPL (R48G) or PkBPL (R48S), referred to as PkBPL; MfBPL R40 amino acid residue mutation: R40G, MfBPL (R40G), is referred to as MfBPL.

The coding nucleotide sequences of PhBPL (R48G), PkBPL (R48G) and MfBPL (R40G) are shown in SEQ ID NO. 4-6 in sequence.

SEQ ID NO.1：

MLGLKTSIIGRRVIYFQEITSTNEFAKTSYLEEGTVIVADKQTMGHGRLNRKWESPEGGLWLSIVLSPKVPQKDLPKIVFLGAVGVVETLKEFSIDGRIKWPNDVLVNYKKIAGVLVEGKGDKIVLGIGLNVNNKVPNGATSMKLELGSEVPLLSVFRSLITNLDRLYLNFLKNPMDILNLVRDNMILGVRVKILGDGSFEGIAEDIDDFGRLIIRLDSGEVKKVIYGDVSLRFL；

SEQ ID NO.2：

MLSLKTSIIGKKVIYYQEISSTNDVAKSLDVEEGTVIVADRQTKGRGRLNRKWISPEGGLWLSVVLKPKVAPQDIPKIVFLGAIGVVRTLEELSIPGRIKWPNDVLVNFRKISGILTEKVGEKVILGIGINVNNNTPENGIAVKNVLGKEVSLVHVFKILLENLDELYEIYLKSPGTIVELARELMILNVPVKVLGNGEVVGIAEDIDEDGRLVLRLGNGEIRKIIYGDVSLRFL；

SEQ ID NO.3：

MEIIHLSEVDSTNEYAKKLAKEGKRNFVVLADKQSTGKGRWGRVWYSDEGGLYFTIVLDSNEYDPKVINLITPISIIETLKNYTDKELGIKFPNDIMVKVNGDYKKLGGILAELINGYMIIGIGINVNNPIRKEIREIAVSLKEVVGKEIDRVEIFNDFLKRFEDYLKKLKNNEIDDYEILKNYKKYSITIGRTVKILLSNNEVITGKVYDIDFDGIVLGTEEGIEKIPTGICIHVR；

SEQ ID NO.4

atgctgggcctgaagacaagcatcatcggccggcgggtcatctacttccaggagatcaccagcaccaacgagttcgccaagaccagctacctggaggagggaaccgtgatcgtggccgataagcagaccatgggccacggaggcctgaacagaaagtgggagtccccagaaggaggcctgtggctgtctatcgtgctcagccctaaagtgccccagaaggacctgcctaagatcgtgttcctgggagcagtgggcgtggtggaaaccctgaaggagttcagcatcgacggcagaatcaagtggcccaacgacgtgctcgtgaactacaagaagatcgccggcgtgctggtcgagggcaaaggcgacaagatcgtgctgggcatcggcctgaacgtgaacaacaaggtgcccaacggcgccacaagcatgaaactggagctgggatcagaagtgcctctgctgagcgtgttcaggagcctgatcaccaacctggaccggctgtacctgaacttcctgaagaaccccatggacatcctgaacctcgtgcgggacaacatgatcctgggcgtgagagtgaagatcctgggcgacggcagcttcgagggcatcgccgaggatatcgacgacttcgggcggctgatcatcaggctggacagcggcgaggtcaagaaggtcatctacggcgacgtgtccctgagattcctgtag；

SEQ ID NO.5：

atgctgagcctgaagaccagcatcatcggcaagaaggtcatctactaccaggagatcagcagcaccaacgacgtggctaagagcctggacgtggaggaaggaaccgtgatcgtggccgataggcagaccaagggaaggggaggcctgaatcgcaagtggatcagcccagaaggaggactctggctgtcagtggtgctcaagcctaaagtggcccctcaggacatccccaagatcgtgtttctgggcgccatcggcgtcgtgagaacactggaggagctgagcatccccggcagaatcaagtggcccaacgacgtgctcgtgaacttccggaagatcagcggcatcctgaccgagaaggtcggcgagaaggtcatcctgggcatcggcatcaacgtgaacaacaacacccccgagaacggcattgccgtgaagaacgtgctgggcaaggaagtgtctctggtgcacgtgttcaagatcctgctggagaacctggacgagctgtacgagatctacctgaagagccccggcaccatcgtggaactggccagggagctgatgatcctgaacgtgcccgtgaaggtgctgggaaacggagaggtcgtgggaatcgccgaggatatcgacgaggacggcagactggtgctgagactgggaaacggcgagatccggaagatcatctacggcgacgtgtccctgagattcctgtag；

SEQ ID NO.6：

atggagatcatccacctgagcgaagtggacagcaccaacgagtacgccaagaagctggccaaggagggcaagcggaacttcgtggtgctggccgacaagcagagcacaggaaaaggcggctggggcagagtgtggtatagcgacgagggaggcctgtacttcaccatcgtgctggacagcaacgagtacgaccccaaggtcatcaacctgatcacccccatcagcatcatcgagaccctgaagaactacaccgacaaggagctgggcatcaagttccccaacgacatcatggtgaaggtcaacggcgactacaagaagctgggcggaatcctggccgaactgatcaacggctacatgatcatcggcatcggcatcaacgtgaacaaccccatccggaaggagatccgggagatcgcagtgtccctgaaggaggtcgtgggaaaggagatcgaccgggtggagatcttcaacgacttcctgaagcgcttcgaggactacctgaagaagctgaagaacaacgagatcgacgactacgagatcctgaagaactacaagaagtacagcatcaccatcggccggaccgtgaagatcctgctgagcaacaacgaggtcatcaccggcaaggtgtacgacatcgacttcgacggcatcgtgctgggaacagaggagggcatcgagaagatccccaccggcatttgcatccacgtccgatag。

Example 2 construction and expression of recombinant plasmid for protein biotin ligase BPL

(1) Construction of recombinant plasmid

The eukaryotic expression vector pcDNA3.1(-) is subjected to double enzyme digestion by BamHI and HindIII and then recovered, and then is connected with a novel biotin ligase BPL (PhBPL, PkBPL and MfBPL) PCR fragment (SEQ ID NO. 4-6) which is recovered after double enzyme digestion by BamHI and HindIII under the action of T4 ligase, DH5 alpha competent cells are transformed and then coated on an LB plate containing benzyl, after overnight culture, single colony amplification culture is selected, plasmids are extracted, sequencing identification is carried out, and the identified positive plasmids are the required recombinant plasmids.

(2) Transient expression of novel biotin ligase by mammalian cells

In order to construct a mammalian cell line expressing the novel biotin ligase, the pcDNA3.1 recombinant of step (1) was transfected into 293T cells using Lipofectamine 2000 transfection method, the specific steps were as follows:

one day before transfection, 293T cells were seeded in six-well plates at 37 ℃ with 5% CO₂And culturing in a DMEM complete culture medium containing 10% fetal calf serum until the cell density is 40% -60% during transfection. Mu.g of recombinant plasmid was diluted in 250. mu.L of Opti-MEM serum-reduced medium and gently mixed. Another 10. mu.L of Lipofectamine 2000 was diluted in 250. mu.L of Opti-MEM reduced serum medium. The mixture was allowed to stand at room temperature for 5 minutes. Mix dilutions of Lipofectamine 2000 and linearized plasmid. Gently mixed and left to stand at room temperature for 20 minutes. Adding 500 μ L of mixed solution into the wells for inoculating 293T cells, gently shaking the cell plate back and forth to mix the mixed solution with the culture solution in the wells, transferring into CO₂Culturing in an incubator. The next day, culture was continued by replacing fresh DMEM complete medium containing 10% fetal bovine serum.

(3) Detection of biotin labeling by Western blotting

Cells were harvested 24h after transfection and labeled by Western blot analysis, as shown in FIG. 1 (left panel, right panel, statistics), PhBPL, PkBPL and MfBPL showed labeling activity similar to that of published AaBPL (BioID 2).

The Western blotting method comprises the following specific steps:

(a) preparation of SDS-PAGE gel

Firstly, selecting a long glass plate (1mm or 0.75mm) with proper thickness, aligning with a short glass plate, clamping on a glue making clamp, and filling ddH into a gap between two plates by using a wash bottle₂O, performing a leak test, and if the horizontal plane does not drop for one to two minutes, indicating that the two rubber plates are tightly matched, pouring water out and sucking the water by using absorbent paper. A piece of 10% separation gel was prepared from a clean beaker according to the following table 1:

TABLE 1 separation gel formulation ingredients

Pouring the separation gel solution into the gap between the two plates, and adding ddH to the gel layer along the glass plate wall by using a wash bottle₂O, after about 20min, the gelatin can be obviously layered with water, namely the gelatin is solidified;

the supernatant was poured out and blotted dry with absorbent paper, a comb (10 or 15 holes) corresponding to the thickness of the slab was prepared, and concentrated gum was prepared as in table 2 below:

TABLE 2 concentrated gel formulation ingredients

The concentrated gum solution was carefully added to the upper layer of the separation gel to avoid the formation of bubbles as much as possible. After the rubber plate is filled, carefully insert the comb, and after about 10min, the edge of the comb teeth is clearly outlined, that is, the gelatin is solidified. And taking the rubber plate out of the rubber clamp, and washing the rubber plate with water for later use.

(b) SDS-PAGE gel electrophoresis

And (3) installing the glue and the rubber plate on an electrophoresis tank, filling electrophoresis buffer solution into the inner tank, and adding the outer tank to the indication scale. The prepared protein sample is dripped into the glue hole, then the cover is covered to connect with a power supply, and a two-step electrophoresis procedure is set: 80V15min, 150V 45min, stop when bromophenol blue indicator tape runs to the lower edge of the glue. Prizing two glass plates under running water, taking out the glue, and soaking in different solutions according to requirements, such as Coomassie brilliant blue dye solution, silver nitrate dye fixing solution or film transfer buffer solution.

(c) Film transfer

An NC membrane having a size similar to that of the gel was prepared, and filter paper was prepared, and similarly soaked in a pre-cooled membrane-transfer buffer. Stacking the sponge, the filter paper, the NC membrane, the glue, the filter paper and the sponge in sequence (no air bubbles should exist between the glue and the membrane), clamping the laminated sponge and the NC membrane by a membrane rotating clamp, installing the laminated sponge and the NC membrane in a membrane rotating groove, and filling the membrane rotating solution. The film transferring groove is placed in an ice bath or a 4-degree refrigerator, and the film is transferred for 1 hour by 200-250 mA.

(d) Protein signal detection

Placing the NC membrane after the membrane transfer into a confining liquid, shaking for 1h, transferring to diluted Streptavidin-HRP, incubating for 1h or overnight at 4 degrees, rinsing the membrane in a TBST solution for 3 times after the incubation, and adopting Luminata^TMForte developed as a substrate and photographed in a BioRad ChemiDoc XRS + gel imaging system.

Alternatively, blocked NC membranes were incubated with diluted Streptavidin-Alexa Fluor 680 for 1h, after which the membranes were rinsed 3 times in TBST solution. The protein band signals were detected and recorded using an Odyssey two-color infrared excitation light imaging system.

Example 3 conditional exploration of proximity proteins labelled with protein biotin ligase BPL

Respectively recombining and cloning novel biotin ligase BPL (PhBPL, PkBPL and MfBPL) (SEQ ID NO. 4-6) and telomere binding protein TRF1 to a lentiviral vector, adding HA antigen at the N end of the lentiviral vector to form a pLenti-HA-BPL-TRF 1 vector, and simultaneously constructing a pLenti-HA-BPL-NLS vector.

HEK293T cells were transferred to 6cm²And (4) performing transfection when the density of the culture dish reaches 70% -80%. Before transfection, the following transfection systems shown in Table 3 were prepared as required:

TABLE 3 transfection System

After the system is prepared, the mixture is blown and uniformly mixed by a liquid transfer gun and is kept stand for 15min at room temperature. Adding into cultured cell culture medium, shaking front and back, and mixing; after 6 hours, the fresh culture medium is replaced, and the culture is continued for 48 hours; and collecting the virus liquid supernatant.

After virus collection, virus infection of target cells is performed. HeLa cells were first plated to ensure that the cell concentration was below 60%, polybrene was added to the virus solution at a concentration of 10mg/ml to a final concentration of 8. mu.g/ml, and mixed well. The medium in the target cells is sucked up, the virus mixed solution is added, the fresh medium is added to supplement the virus mixed solution to 5ml, and the cells are statically placed in a 37-degree incubator. And after infection is finished, collecting a sample and carrying out Western blot detection on the expression of the fusion protein.

After confirming that the cells over-express the Biotin ligase-telomere binding protein TRF1 fusion protein, the cells were cultured in a medium without endogenous Biotin, and the following conditions were investigated:

(1) marking time: 0min, 1min, 10min, 30min, 1h, 2h, 4h, 8h and 16 h;

the results are shown in fig. 2-3, and fig. 2 is a graph (fig. 2A) and a statistical graph (fig. 2B) of the detection results of the novel biotinidase BPL by the labeled protein immunoblotting (Western blot) at different times, and the results show that the labeling intensity is stronger as the labeling time is longer, the labeling saturation is approached by PhBPL-TRF 1 at 2h, and the saturation is absent in PkBPL, MfBPL and AaBPL (BioID 2). Fig. 3 is a comparison graph of labeling intensity of novel biotin enzyme PhBPL with nuclear localization signal and BioID2 (left is a detection result graph, right is a statistical graph), and the result shows that the labeling activity of PhBPL-NLS is far stronger than that of AaBPL (BioID2), the PhBPL has a certain effect after being labeled for 10 minutes, and the effect of AaBPL labeling for 16 hours can be achieved after 4 hours.

(2) The biotin concentration: 0. mu.M, 0.5. mu.M, 5. mu.M and 50. mu.M

Figure 4 shows the effect of the novel biotinidase BPL on protein labelling at different biotin concentrations. The results show that the novel biotinidase BPL shows labeling activity at 0.5. mu.M, 5. mu.M and 50. mu.M, with preferred biotin concentrations of 5. mu.M to 50. mu.M.

(3) Streptavidin purification experiment

HeLa cells overexpressing Biotin ligase-TRF 1 were lysed, the protein labeled with Biotin was purified using streptavidin, and a novel Biotin ligase-based labeling system was examined for whether it could label the protein RAP1 in the vicinity of TRF1 (rather than by direct interaction).

The specific implementation flow is as follows:

cell samples were collected (6cm petri dish), lysed using 480. mu.L of RIPA for 15min, and centrifuged at 15,000g at 4 ℃ for 15 min. 400 μ L of the supernatant was incubated with streptavidin-conjugated magnetic beads for 2h at 4 ℃ with three-dimensional rotation. Take 40. mu.L as Input sample. Samples of magnetic beads that have undergone pulldown are washed twice with 1mL of 2% SDS, 500. mu.L of TBST, and 500. mu.L of TBS, respectively. And adding the washed magnetic bead sample into an SDS-containing eluent, boiling for 10 minutes in boiling water, and then carrying out Western blot detection.

The results are shown in fig. 5, indicating that the labeling systems based on PhBPL, PkBPL and MfBPL, like BioID2, all exhibited labeling ability for RAP1, with PhBPL being the most potent label and PkBPL being the more label-specific.

Sequence listing

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<120> a novel protein biotin ligase and proximity labeling system PhastID based thereon

<141> 2021-11-04

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Glu Leu Ile Asn Gly Tyr Met Ile Ile Gly Ile Gly Ile Asn Val Asn

115 120 125

Asn Pro Ile Arg Lys Glu Ile Arg Glu Ile Ala Val Ser Leu Lys Glu

130 135 140

Val Val Gly Lys Glu Ile Asp Arg Val Glu Ile Phe Asn Asp Phe Leu

145 150 155 160

Lys Arg Phe Glu Asp Tyr Leu Lys Lys Leu Lys Asn Asn Glu Ile Asp

165 170 175

Asp Tyr Glu Ile Leu Lys Asn Tyr Lys Lys Tyr Ser Ile Thr Ile Gly

180 185 190

Arg Thr Val Lys Ile Leu Leu Ser Asn Asn Glu Val Ile Thr Gly Lys

195 200 205

Val Tyr Asp Ile Asp Phe Asp Gly Ile Val Leu Gly Thr Glu Glu Gly

210 215 220

Ile Glu Lys Ile Pro Thr Gly Ile Cys Ile His Val Arg

225 230 235

<210> 4

<211> 708

<212> DNA

<213> archaebacteria (Pyrococcus horikoshii)

<400> 4

atgctgggcc tgaagacaag catcatcggc cggcgggtca tctacttcca ggagatcacc 60

agcaccaacg agttcgccaa gaccagctac ctggaggagg gaaccgtgat cgtggccgat 120

aagcagacca tgggccacgg aggcctgaac agaaagtggg agtccccaga aggaggcctg 180

tggctgtcta tcgtgctcag ccctaaagtg ccccagaagg acctgcctaa gatcgtgttc 240

ctgggagcag tgggcgtggt ggaaaccctg aaggagttca gcatcgacgg cagaatcaag 300

tggcccaacg acgtgctcgt gaactacaag aagatcgccg gcgtgctggt cgagggcaaa 360

ggcgacaaga tcgtgctggg catcggcctg aacgtgaaca acaaggtgcc caacggcgcc 420

acaagcatga aactggagct gggatcagaa gtgcctctgc tgagcgtgtt caggagcctg 480

atcaccaacc tggaccggct gtacctgaac ttcctgaaga accccatgga catcctgaac 540

ctcgtgcggg acaacatgat cctgggcgtg agagtgaaga tcctgggcga cggcagcttc 600

gagggcatcg ccgaggatat cgacgacttc gggcggctga tcatcaggct ggacagcggc 660

gaggtcaaga aggtcatcta cggcgacgtg tccctgagat tcctgtag 708

<210> 5

<211> 708

<212> DNA

<213> archaebacteria (Pyrococcus kukukukumulkanii)

<400> 5

atgctgagcc tgaagaccag catcatcggc aagaaggtca tctactacca ggagatcagc 60

agcaccaacg acgtggctaa gagcctggac gtggaggaag gaaccgtgat cgtggccgat 120

aggcagacca agggaagggg aggcctgaat cgcaagtgga tcagcccaga aggaggactc 180

tggctgtcag tggtgctcaa gcctaaagtg gcccctcagg acatccccaa gatcgtgttt 240

ctgggcgcca tcggcgtcgt gagaacactg gaggagctga gcatccccgg cagaatcaag 300

tggcccaacg acgtgctcgt gaacttccgg aagatcagcg gcatcctgac cgagaaggtc 360

ggcgagaagg tcatcctggg catcggcatc aacgtgaaca acaacacccc cgagaacggc 420

attgccgtga agaacgtgct gggcaaggaa gtgtctctgg tgcacgtgtt caagatcctg 480

ctggagaacc tggacgagct gtacgagatc tacctgaaga gccccggcac catcgtggaa 540

ctggccaggg agctgatgat cctgaacgtg cccgtgaagg tgctgggaaa cggagaggtc 600

gtgggaatcg ccgaggatat cgacgaggac ggcagactgg tgctgagact gggaaacggc 660

gagatccgga agatcatcta cggcgacgtg tccctgagat tcctgtag 708

<210> 6

<211> 714

<212> DNA

<213> archaea (Methanocadococcus fervens)

<400> 6

atggagatca tccacctgag cgaagtggac agcaccaacg agtacgccaa gaagctggcc 60

aaggagggca agcggaactt cgtggtgctg gccgacaagc agagcacagg aaaaggcggc 120

tggggcagag tgtggtatag cgacgaggga ggcctgtact tcaccatcgt gctggacagc 180

aacgagtacg accccaaggt catcaacctg atcaccccca tcagcatcat cgagaccctg 240

aagaactaca ccgacaagga gctgggcatc aagttcccca acgacatcat ggtgaaggtc 300

aacggcgact acaagaagct gggcggaatc ctggccgaac tgatcaacgg ctacatgatc 360

atcggcatcg gcatcaacgt gaacaacccc atccggaagg agatccggga gatcgcagtg 420

tccctgaagg aggtcgtggg aaaggagatc gaccgggtgg agatcttcaa cgacttcctg 480

aagcgcttcg aggactacct gaagaagctg aagaacaacg agatcgacga ctacgagatc 540

ctgaagaact acaagaagta cagcatcacc atcggccgga ccgtgaagat cctgctgagc 600

aacaacgagg tcatcaccgg caaggtgtac gacatcgact tcgacggcat cgtgctggga 660

acagaggagg gcatcgagaa gatccccacc ggcatttgca tccacgtccg atag 714

Claims

1. A protein biotin ligase BPL characterized in that,

(a) the amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;

or

2. A mutein biotin ligase BPL characterized by mutations in the amino acid sequence R48G or R48S of SEQ ID No. 1; or the amino acid sequence R48G or R48S mutation described in SEQ ID NO. 2; or the amino acid sequence R40G mutation described in SEQ ID NO. 3.

3. A nucleotide sequence encoding the mutein biotin ligase BPL according to claim 2.

4. A recombinant expression vector comprising a mutein biotin ligase BPL encoding nucleotide sequence according to claim 3.

5. A host cell comprising the recombinant expression vector of claim 4.

6. Use of the protein biotin ligase BPL according to claim 1 or the mutein biotin ligase BPL according to claim 2 for the labeling of adjacent proteins or for the preparation of a system for labeling adjacent proteins.

7. A proximal protein labeling system comprising the protein biotin ligase BPL according to claim 1 or the mutein biotin ligase BPL according to claim 2.

8. A method for labeling adjacent protein, which is characterized in that the target protein to be researched and mutant protein biotin ligase BPL form fusion protein, the fusion protein is labeled by biotin, the protein labeled by biotin ligase around the target protein is purified by a biotin-avidin system, and the protein having interaction with the protein is further identified by a high-throughput method.

9. The method according to claim 8, wherein the biotin concentration is 0.5 μ M to 50 μ M.

10. The method of claim 8, wherein the labeling time is 10min to 16 h.