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

A biotin ligase, protein labeling technology, applied in the field of adjacent labeling systems, can solve the problems of weak labeling ability, long time required, large molecular weight of enzyme protein, etc., and achieve short labeling time, high labeling intensity and great application prospects. Effect

Pending Publication Date: 2022-03-08
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these proximity labeling techniques have certain defects, such as: the labeling ability is weak, so it takes a long time, the ability to detect transient or weakly interacting proteins is weak; or the protein molecular weight of the enzyme is large

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining of protein biotin ligase BPL* (PhBPL*, PkBPL* and MfBPL*)

[0040] Obtain biotin ligase sequences of various species on NCBI, including the BPL derived from the archaea Pyrococcushorikoshii, called PhBPL, whose amino acid sequence is shown in SEQ ID NO.1; the BPL derived from the archaea Pyrococcus kukulkanii, called PkBPL, whose amino acid sequence is shown in SEQ ID NO.2; and the BPL derived from the archaea Methanocaldoccus fervens, called MfBPL, whose amino acid sequence is shown in SEQ ID NO.3; analyze its GNGR motif (N represents a basic amino acid ), and mutate the above protein into GNGG form; PhBPL R48 amino acid residue mutation: R48G or R48S mutation, namely PhBPL (R48G) or PhBPL (R48S), called PhBPL*; PkBPL R48 amino acid residue mutation: R48G or R48S mutation , that is, PkBPL (R48G) or PkBPL (R48S), called PkBPL*; MfBPL R40 amino acid residue mutation: R40G, that is, MfBPL (R40G), called MfBPL*.

[0041] The coding nucleotide sequences...

Embodiment 2

[0054] Example 2 Construction and expression of protein biotin ligase BPL* recombinant plasmid

[0055] (1) Construction of recombinant plasmids

[0056] The eukaryotic expression vector pcDNA3.1(-) was recovered after double digestion with BamHI and HindIII, and then combined with the novel biotin ligase BPL* (PhBPL*, PkBPL* and MfBPL*) recovered after double digestion with BamHI and HindIII PCR fragments (SEQID NO.4~6) were ligated under the action of T4 ligase, transformed into DH5α competent cells, spread on LB plates containing ampicillin, picked single colonies after overnight culture, and extracted plasmids for sequencing identification , the identified positive plasmid is the desired recombinant plasmid.

[0057](2) Transient expression of novel biotin ligase in mammalian cells

[0058] In order to construct a mammalian cell line expressing a novel biotin ligase, the pcDNA3.1 recombinant plasmid in step (1) was transfected into 293T cells using the Lipofectamine 2000...

Embodiment 3

[0079] Example 3 Exploration of conditions for labeling adjacent proteins with protein biotin ligase BPL*

[0080] The novel biotin ligases BPL* (PhBPL*, PkBPL* and MfBPL*) (SEQ ID NO.4-6) and the telomere-binding protein TRF1 were recombined and cloned into the lentiviral vector respectively, and the HA antigen was added to its N-terminus , forming the pLenti-HA-BPL*-TRF1 vector, and constructing the pLenti-HA-BPL*-NLS vector at the same time.

[0081] Spread HEK293T cells to 6cm 2 Petri dish, replace its density to 70%-80% for transfection. Before transfection, select and prepare the following transfection systems shown in Table 3 as needed:

[0082] Table 3 Transfection system

[0083]

[0084] After the system is prepared, blow and mix with a pipette gun, and let it stand at room temperature for 15 minutes. Add it to the cultured cell culture medium, shake it back and forth to mix well; change the fresh medium after 6 hours, and continue to cultivate until 48 hours;...

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Abstract

The invention discloses a novel protein biotin ligase and a proximity labeling system PhastID based on the novel protein biotin ligase. The invention specifically discloses a protein biotin ligase BPL, a modified mutant protein biotin ligase BPL * and an adjacent protein labeling system PhastID based on the biotin ligase BPL or BPL *. Compared with a traditional BioID proximity labeling system, the labeling system containing the protein biotin ligase BPL * has the advantages that the labeling intensity is higher, the required labeling time is shorter, and the application prospect is larger.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a novel protein biotin ligase BPL, a mutated protein biotin ligase fusion protein BPL* and a proximity labeling system PhastID based on the protein biotin ligase BPL or BPL*. Background technique [0002] Protein biotin ligase can activate biotin (Biotin) to form biotinyl-5'-adenylate Biotinyl-5'-AMP, and label biotin to its substrate. After artificial modification, the ligase can lose the specific labeling of the substrate, and the released Biotinyl-5'-AMP is chemically unstable, and can randomly react with adjacent lysine residues to make it covalently labeled For biotin, this range is within about 10nm. Combined with the high affinity and stability between biotin and streptavidin, the protein can be traced, purified and other operations. Since its labeling radius is about 10nm, which is just the premise of protein-protein interaction (Protein-Protein Inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/85C12N5/10C12N15/867C12N15/62C07K1/13
CPCC12N9/93C12N15/85C12N5/0686C12N15/86C12N15/62C07K1/13C12Y603/04015C12N2800/107C12N2510/02C12N2740/15043C07K2319/00
Inventor 松阳洲冯然刘峰吴苏李克成李影影周志芬李若菲刘熠
Owner SUN YAT SEN UNIV
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