CN-121995054-A - Method for identifying p97 interaction protein based on proximity labeling technology

Abstract

The invention belongs to the technical field of biology, relates to a method for identifying p97 interaction proteins based on a proximity labeling technology, establishes a method for identifying p97 interaction proteins based on a Tet-on induced TurboID proximity labeling technology, avoids the possible high background and nonspecific labeling problems of transient transformation, and discovers various proteins interacting with p97 proteins in vivo, including proteins which are difficult to discover in vivo transient and weak interactions by a general method. The detection method has high detection rate and sensitivity, lays a foundation for subsequent p97 biological function research, and has a certain application prospect.

Inventors

• LI JINGHONG
• LI XU
• LI CHUNTONG
• WANG HAN

Assignees

• 青岛大学

Dates

Publication Date

20260508

Application Date

20260213

Claims (5)

1. 1. A method for identifying p97 interaction protein based on proximity labeling technology, which is characterized by comprising the following specific steps: (1) Construction of Tet-on-TurboID-p 97-FLAG-inducible lentiviral expression vector PTRIPZ is taken as a slow virus vector, turboID-p97-FLAG sequence is connected to the slow virus vector through a homologous recombination mode, then bacteria are transformed, the bacteria are cultured, plasmids are extracted and sequenced, and the nucleotide sequence of TurboID-p97-FLAG is shown as SEQ ID NO. 1; (2) Co-transfecting a lentiviral vector with a Tet-on-TurboID-p97-FLAG plasmid and an auxiliary plasmid pSPAX and pMD2G which are accurate in sequencing into 293T cells, culturing for about 48-72 hours, collecting a supernatant culture solution containing viruses, concentrating the viruses through ultra-high speed centrifugation, and re-suspending by using PBS buffer solution to obtain a lentiviral supernatant with infectious capacity; (3) Infecting 293T cells with lentivirus supernatant with infectivity, adding 1 mug/ml puromycin after 24 hours of infection, continuously screening to remove uninfected cells until a Tet-on_ TurboID-p97-FLAG stable recombinant cell line is obtained by screening, and freezing the cells at-80 ℃ for later use after expanding culture; (4) Culturing Tet-on_ TurboID-p97-FLAG recombinant cells, adding 1 mug/mL tetracyclomycin 24 hours before stimulation to induce turboID-p97-FLAG expression, then adding biotin with a final concentration of 50 mu M to incubate 30min, lysing the incubated recombinant cells, enriching streptavidin magnetic beads, and carrying out mass spectrum identification.
2. 2. A recombinant plasmid is characterized in that the recombinant plasmid is TurboID-p97-FLAG, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 1.
3. 3. A recombinant lentivirus comprising the recombinant plasmid of claim 2, wherein the lentivirus is pTRIPZ.
4. 4. A recombinant cell, wherein the recombinant cell is a cell infected with the recombinant lentivirus of claim 3.
5. 5. Use of the recombinant plasmid of claim 2 or the recombinant lentivirus of claim 3 for detecting the interaction of p97 protein with other proteins.

Description

Method for identifying p97 interaction protein based on proximity labeling technology Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for identifying p97 interaction protein based on a proximity labeling technology. Background VCP (also called p 97) is a homohexamer AAA+ATPase, is an important conservative member of the ATP (ATPase) AAA family, is a key medium of protein steady-state mechanism in eukaryotic cells, is a key regulator of endoplasmic reticulum related degradation, and p97 plays a role in biological process and depends on a large amount of interaction proteins, so that searching for the interaction proteins lays a foundation for subsequent biological function research of p97, and has a certain application prospect. Most experiments, based on Molecular & Cellular Proteomics 2016, in literature "Valosin-containing protein (VCP)–Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor"（ Raymond J. Deshaies et al, studied the p97 interacting protein by immunoprecipitation mass spectrometry (Immunoprecipitation-Mass Spectrometry, IP-MS). However, p97 was removed from the living cell environment in immunoprecipitation mass spectrometry and many weak transient interactions were not captured. The rapid development of proximity-labeled mass spectrometry (Proximity Labeling Mass Spectrometry, PL-MS) provides a powerful tool for exploring the spatial distribution and interaction network of intracellular proteins. Classical PL methods such as BioID, APEX and TurboID rely on catalytic enzymes to generate short lived active intermediates to covalently label adjacent proteins. The development of proximity labelling technology solves the above drawbacks of conventional IP-MS searching for interacting proteins, and can capture transient weak protein interactions in living cells. Currently, the proximity technology mainly comprises BioID, turboID and APEX, wherein both BioID and APEX have certain limitations. BioID labeling is long (incubation for 8-24 hours) and may capture non-physiological interactions. APEX or APEX2 relies on H 2O2 and is highly cytotoxic and unsuitable for organoid or in vivo studies. Compared with TurboID technology, the marking speed and the safety are both considered, the marking time is shortened by 10 minutes, H 2O2 is not needed, and the toxicity problem is thoroughly solved. Disclosure of Invention The object of the present invention is to provide a method for identifying p97 interacting proteins based on proximity labelling technology. In order to achieve the above object, the present invention provides a method for identifying p97 interacting protein based on proximity labeling technology, comprising the following specific steps: (1) Construction of Tet-on-TurboID-p 97-FLAG-inducible lentiviral expression vector PTRIPZ is taken as a slow virus vector, turboID-p97-FLAG sequence is connected to the slow virus vector through a homologous recombination mode, then bacteria are transformed, the bacteria are cultured, plasmids are extracted and sequenced, and the nucleotide sequence of TurboID-p97-FLAG is shown as SEQ ID NO. 1; (2) Co-transfecting a lentiviral vector with a Tet-on-TurboID-p97-FLAG plasmid and an auxiliary plasmid pSPAX and pMD2G which are accurate in sequencing into 293T cells, culturing for about 48-72 hours, collecting a supernatant culture solution containing viruses, concentrating the viruses through ultra-high speed centrifugation, and re-suspending by using PBS buffer solution to obtain a lentiviral supernatant with infectious capacity; (3) Infecting 293T cells with lentivirus supernatant with infectivity, adding 1 mug/ml puromycin after 24 hours of infection, continuously screening to remove uninfected cells until a Tet-on_ TurboID-p97-FLAG stable recombinant cell line is obtained by screening, and freezing the cells at-80 ℃ for later use after expanding culture; (4) Culturing Tet-on_ TurboID-p97-FLAG recombinant cells, adding 1 mug/mL tetracyclomycin 24 hours before stimulation to induce turboID-p97-FLAG expression, then adding biotin with a final concentration of 50 mu M to incubate 30min, lysing the incubated recombinant cells, enriching streptavidin magnetic beads, and carrying out mass spectrum identification. The invention also provides a recombinant plasmid, which is turboID-p97-FLAG, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 1. The invention also provides a recombinant lentivirus, which comprises the recombinant plasmid, wherein the lentivirus is pTRIPZ. The invention also provides a recombinant cell, which is a cell infected by the recombinant lentivirus. The invention also provides the application of the recombinant plasmid and the recombinant lentivirus in detecting the interaction of the p97 protein and other proteins. Compared with the prior art, the invention has the beneficial effects that a method for iden