Modifications and targeting of protein termini. Part B /

Detalles Bibliográficos
Clasificación:Libro Electrónico
Formato: Electrónico eBook
Idioma:Inglés
Publicado: [S.l.] : Academic Press, 2023.
Colección:Methods in enzymology ; v. 686
Temas:
Proteins.
Enzymology.
Acceso en línea:Texto completo
Tabla de Contenidos:
  • Chapter Five: In vitro production of N-degron fused proteins and its application
  • 1. Introduction
  • 2. Key resources
  • 2.1. Key resources table
  • 2.2. Materials
  • 2.3. Buffer recipes
  • 3. Preparation of N-degron attached ZZ-domain of p62/SQSTM1
  • 3.1. Molecular cloning LC3B fused human p62 ZZ-domain
  • 3.1.1. PCR amplification of ZZ-p62 (126-180)
  • 3.1.2. Digestion of insert PCR product
  • 3.1.3. Digestion of the pET-His-LC3B vector
  • 3.1.4. Ligation
  • 3.1.5. Transformation
  • 3.1.6. Preparation of plasmids
  • 3.1.7. Notes
  • 3.2. Expression of N-degron fused ZZ-p62
  • 3.2.1. Cell culture
  • 3.2.2. Cell harvest
  • 3.2.3. Cell lysis
  • 3.2.4. Notes
  • 3.3. Purification of N-degron fused ZZ-p62
  • 3.3.1. His-affinity column chromatography
  • 3.3.2. Ion exchange column chromatography
  • 3.3.3. LC3B-tag cleavage and removal
  • 3.3.4. Size exclusion chromatography
  • 4. Preparation of human ATG4B protease
  • 4.1. Purification of human ATG4B
  • 4.1.1. His-affinity column chromatography
  • 4.1.2. Ion exchange column chromatography
  • 5. Applications of N-degron fused ZZ-p62 or N-degron fused proteins of interest
  • 5.1. Crystallization of REEED-ZZ p62
  • 5.2. Notes
  • 5.3. Structure determination
  • 5.4. Other applications of LC3B-fusion technique
  • 6. Conclusions
  • Acknowledgments
  • References
  • Chapter Six: TEV protease cleavage in generation of artificial substrate proteins bearing neo-N-termini
  • 1. Introduction
  • 2. General method and equipment
  • 3. Construction of a self-cleaving fusion protein
  • 3.1. Materials and equipment
  • 3.2. Procedure
  • 3.3. Notes
  • 4. Construction of fusion proteins containing the TEV protease recognition site
  • 4.1. Materials and equipment
  • 4.2. Procedure
  • 4.3. Notes
  • 5. Expression of proteins
  • 5.1. Materials and equipment
  • 5.2. Procedure
  • 5.3. Notes
  • 6. SDS PAGE and western blot.
  • 6.1. Materials and equipment
  • 6.2. Procedure
  • 7. Summary and conclusion
  • Acknowledgments
  • References
  • Chapter Seven: Affinity isolation and biochemical characterization of N-degron ligands using the N-recognin, ClpS
  • 1. Introduction
  • 2. Determine N-degron specificity of ClpS (N-recognin) using immobilized peptide arrays
  • 2.1. Materials and buffer recipes
  • 2.2. Preparation of peptide array (for first use)
  • 2.3. Incubation of the N-recognin (ClpS) with the peptide array and preparation for transfer to PVDF membrane
  • 2.4. Transfer of the bound N-recognin to PVDF membranes
  • 2.5. Visualization and analysis of the transferred N-recognin (on PVDF membrane)
  • 2.6. Regeneration, reuse and storage of peptide arrays
  • 3. ClpS affinity chromatography to isolate (and identify) natural N-degron substrates/ligands
  • 3.1. Materials and buffer recipes
  • 3.2. Preparation and equilibration of Ni-NTA agarose beads
  • 3.3. Bind ClpS-His10 (WT and mutant) to Ni-NTA agarose
  • 3.4. Affinity purification of N-degron ligands from a bacterial lysate (all steps are performed at 4C)
  • References
  • Chapter Eight: Monitoring the interactions between N-degrons and N-recognins of the Arg/N-degron pathway
  • 1. Introduction
  • 2. Preparation of N-degrons for pulldown assays
  • 2.1. X-peptides carrying type 1 or type 2N-degrons
  • 2.1.1. Equipment
  • 2.1.2. Buffers and reagents
  • 2.1.3. Procedure
  • 2.1.4. Notes
  • 2.2. Biotin-conjugated chemical mimics of the N-degrons
  • 2.2.1. Equipment
  • 2.2.2. Buffers and reagents
  • 2.2.3. Procedure
  • 2.2.4. Notes
  • 3. Preparations of N-recognins for pulldown assays
  • 3.1. Extracts containing N-recognins in cultured cells
  • 3.1.1. Equipment
  • 3.1.2. Buffers and reagents
  • 3.1.3. Procedure
  • 3.1.4. Notes
  • 3.2. Extracts containing N-recognins from murine tissues
  • 3.2.1. Equipment.
  • 3.2.2. Buffers and reagents
  • 3.2.3. Procedure
  • 3.2.4. Notes
  • 3.3. Purification of N-recognins expressed in bacteria
  • 3.3.1. Equipment
  • 3.3.2. Buffers and reagents
  • 3.3.3. Procedure
  • 3.3.4. Notes
  • 4. N-degron pulldown assays
  • 4.1. Pulldown assays using crude lysates
  • 4.1.1. Equipment
  • 4.1.2. Buffers and reagents
  • 4.1.3. Procedure
  • 4.1.4. Notes
  • 4.2. Pulldown assays with purified proteins
  • 4.2.1. Equipment
  • 4.2.2. Buffers and reagents
  • 4.2.3. Procedure
  • 4.2.4. Notes
  • 4.3. Competitive inhibition of N-degron pulldown assays
  • 4.3.1. Equipment
  • 4.3.2. Buffers and reagents
  • 4.3.3. Procedure
  • 4.3.4. Notes
  • 5. Monitoring the proteome captured by N-degrons
  • 5.1. Immunoblotting analysis
  • 5.1.1. Equipment
  • 5.1.2. Buffers and reagents
  • 5.1.3. Procedure
  • 5.1.4. Notes
  • 5.2. Liquid chromatography-mass spectrometry (LC/MS) analysis
  • 5.2.1. Equipment
  • 5.2.2. Buffers and reagents
  • 5.2.3. Procedure
  • 5.2.4. Notes
  • 6. Pulldown of N-degron substrates using ubiquitin-fusion technique (UFT)
  • 6.1. Equipment
  • 6.2. Buffers and reagents
  • 6.3. Procedure
  • 6.4. Notes
  • 7. Summary and conclusions
  • Acknowledgments
  • References
  • Chapter Nine: In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway
  • 1. Introduction
  • 2. General method and equipment
  • 3. Design of constructs
  • 4. Recombinant protein expression of E3 ligase
  • 4.1. Materials and equipment
  • 4.2. Procedure
  • 4.3. Notes
  • 5. Protein purification after recombinant expression via IMAC
  • 5.1. Materials and equipment
  • 5.2. Procedure
  • 5.3. Notes
  • 6. In vitro autoubiquitination assay
  • 6.1. Materials and equipment
  • 6.2. Procedure
  • 6.3. Notes
  • 7. Western blot analyses
  • 7.1. Materials and equipment
  • 7.2. Procedure
  • 7.3. Notes
  • Acknowledgments
  • References.

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