Title:
SIRT6 H133Y PROTEIN, METHOD FOR ENRICHING MYRISTOYLATION-MODIFIED PEPTIDE FRAGMENTS USING SAME, AND USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2024/021228
Kind Code:
A1
Abstract:
The present invention provides an SIRT6 H133Y protein, a method for enriching myristoylation modified-peptide fragments using same, and use thereof. A method for preparing the protein comprises: taking SIRT6-FLAG as a template to obtain an SIRT6-FLAG fragment, taking a plasmid pET28a as a template to obtain a plasmid fragment, splicing the two fragments to obtain an expression plasmid pET28TEV-SIRT6, and carrying out a PCR site-directed mutagenesis by means of taking the expression plasmid as a template to obtain an overexpression plasmid pET28TEV-SIRT6H133Y, and transfecting the overexpression plasmid into Escherichia coli to purify the protein. According to the present invention, on the basis of the binding force of the SIRT6 H133Y protein to myristoyl groups and by means of immune coprecipitation of myristoylation-modified peptide fragments by His-tag and Flag-tag on the SIRT6 H133Y protein, the myristoylation-modified peptide fragments are obtained by means of enriching, and a myristoylation-modified target protein and a modification site are obtained by means of mass spectrometry detection and analysis.
Inventors:
LI HUA (CN)
LI PENG (CN)
WU NA (CN)
GE JUNBO (CN)
LI PENG (CN)
WU NA (CN)
GE JUNBO (CN)
Application Number:
PCT/CN2022/117402
Publication Date:
February 01, 2024
Filing Date:
September 06, 2022
Export Citation:
Assignee:
ZHONGSHAN HOSPITAL FUDAN UNIV (CN)
International Classes:
C12N9/80; C07K1/107; C12N9/10; C12N15/70; C12P21/06; G01N30/72
Domestic Patent References:
| WO2021179214A1 | 2021-09-16 |
Foreign References:
| CN105087619A | 2015-11-25 | |||
| CN105713957A | 2016-06-29 |
Other References:
GRIMLEY RACHEL, POLYAKOVA OXANA, VAMATHEVAN JESSICA, MCKENARY JOANNE, HAYES BRIAN, PATEL CHAMPA, SMITH JANET, BRIDGES ANGELA, FOSB: "Over Expression of Wild Type or a Catalytically Dead Mutant of SIRTUIN 6 Does Not Influence NFκB Responses", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 7, no. 7, US , pages e39847, XP093133664, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0039847
MARK A. KLEIN, CAN LIU, VYACHESLAV I. KUZNETSOV, JOHN B. FELTENBERGER, WEIPING TANG, JOHN M. DENU: "Mechanism of activation for the sirtuin 6 protein deacylase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 295, no. 5, 31 January 2020 (2020-01-31), US , pages 1385 - 1399, XP055771341, ISSN: 0021-9258, DOI: 10.1074/jbc.RA119.011285
LISZT GREGORY, FORD ETHAN, KURTEV MARTIN, GUARENTE LEONARD: "Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 280, no. 22, 1 June 2005 (2005-06-01), US , pages 21313 - 21320, XP093133668, ISSN: 0021-9258, DOI: 10.1074/jbc.M413296200
MARK A. KLEIN, CAN LIU, VYACHESLAV I. KUZNETSOV, JOHN B. FELTENBERGER, WEIPING TANG, JOHN M. DENU: "Mechanism of activation for the sirtuin 6 protein deacylase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 295, no. 5, 31 January 2020 (2020-01-31), US , pages 1385 - 1399, XP055771341, ISSN: 0021-9258, DOI: 10.1074/jbc.RA119.011285
LISZT GREGORY, FORD ETHAN, KURTEV MARTIN, GUARENTE LEONARD: "Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 280, no. 22, 1 June 2005 (2005-06-01), US , pages 21313 - 21320, XP093133668, ISSN: 0021-9258, DOI: 10.1074/jbc.M413296200
Attorney, Agent or Firm:
SHANGHAI BRIDGE INTELLECTUAL PROPERTY AGENCE CO., LTD. (CN)
Download PDF: