Recombinant Human COP1 protein, His-tagged

• Cat.No.: COP1-11454H
• Product Overview: Recombinant Human COP1 protein(1-349 aa), fused with His tag, was expressed in E.coli.

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Protein Length: 1-349 aa
• Form: The purified protein was Lyophilized from sterile PBS (58mM Na2HPO4,17mM NaH2PO4, 68mM NaCl, pH7.4). 5 % trehalose and 5 % mannitol are added as protectant before lyophilization. The elution buffer contain 300mM imidazole.
• AA Sequence: SRTASQLDEFQECLSKFTRYNSVRPLATLSYASDLYNGSSIVSSIEFDRDCDYFAIAGVTKKIKVYEYDTVIQDAVDIHYPENEMTCNSKISCISWSSYHKNLLASSDYEGTVILWDGFTGQRSKVYQEHEKRCWSVDFNLMDPKLLASGSDDAKVKLWSTNLDNSVASIEAKANVCCVKFSPSSRYHLAFGCADHCVHYYDLRNTKQPIMVFKGHRKAVSYAKFVSGEEIVSASTDSQLKLWNVGKPYCLRSFKGHINEKNFVGLASNGDYIACGSENNSLYLYYKGLSKTLLTFKFDTVKSVLDKDRKEDDTNEFVSAVCWRALPDGESNVLIAANSQGTIKVLELV
• Storage: Short-term storage: Store at 2-8°C for (1-2 weeks).
  Long-term storage: Aliquot and store at -20°C to -80°C for up to 3 months, buffer containing 50% glycerol is recommended for reconstitution. Avoid repeat freeze-thaw cycles.
• Reconstitution: Reconstitute at 0.25 µg/μl in 200 μl sterile water for short-term storage.
  Reconstitution with 200 μl 50% glycerol solution is recommended for longer term storage.

Citation

Imaging and kinetics of the bimolecular complex formed by the tumor suppressor p53 with ubiquitin ligase COP1 as studied by atomic force microscopy and surface plasmon resonance

Journal: International Journal of Nanomedicine    PubMed ID: 29379285    Data: 2022/12/14

Authors: Ilaria Moscetti, Anna Rita Bizzarri, Salvatore Cannistraro

Article Snippet:Recombinant human full-length p53 (43.6 kDa) (hereafter p53) was purchased from Genscript (Piscataway, NJ, USA) by using the BacPower? Guaranteed Bacterial Protein Expression Service.Recombinant human full-length p53 (43.6 kDa) (hereafter p53) was purchased from Genscript (Piscataway, NJ, USA) by using the BacPower? Guaranteed Bacterial Protein Expression Service.. Recombinant human COP1 protein MYC/DDK-tagged (77.5 kDa) (hereafter COP1), also known as RING finger and WD repeat domain 2, was purchased from Creative BioMart (Shirley, NY, USA).. Glutathione S-transferase (GST) (26 kDa) was purchased from GE Healthcare UK Ltd (Little Chalfont, UK).Glutathione S-transferase (GST) (26 kDa) was purchased from GE Healthcare UK Ltd (Little Chalfont, UK).

Schematic representation of the surface chemistry used to covalently bind p53 and COP1 to AFM tips and substrate, respectively. Notes: ( A ) p53 protein was linked to the AFM tip through the ?NH 2 groups of lysines exposed on the protein surface after the tip functionalization with MPTMS and NHS-PEG-MAL crosslinker. ( B ) COP1 protein was immobilized over the aldehyde-functionalized glass surface by randomly targeting amino groups of lysine residues exposed on the protein surfaces. Abbreviations: AFM, atomic force microscopy; COP1, constitutive photomorphogenesis protein 1; MPTMS, 3-mercatopropyl-trimethoxysilane; NHS-PEG-MAL, N -hydroxysuccinimide-polyethyleneglycol-maleimide.

A typical approach–retraction cycle of the p53-functionalized tip over the COP1-functionalized substrate showing a specific unbinding event. Notes: (1) The tip moves toward the substrate. (2) The tip reaches the contact point. (3) A further pressure toward the substrate causes an upward deflection of the cantilever. (4) During the retraction, the cantilever bends downward due to the attractive interaction force of the p53-COP1 complex. (5) The cantilever jumps off, returning to its initial position. Abbreviation: COP1, constitutive photomorphogenesis protein 1.

TM-AFM images recorded in air of COP1, p53, and preincubated p53–COP1 molecules adsorbed on a mica substrate. Notes: ( A ) COP1 sample displaying isolated single spots; inset: cross-section profile of the spot indicated by the white arrow. ( B ) p53 sample showing single (yellow arrow) and bimolecular spots (green arrow); yellow inset: cross-section profile of the single spot and green inset: cross-section profile of the bimolecular spot. ( C ) p53–COP1 sample showing bimolecular complexes; inset: cross-section profile of the complex indicated by the white arrow. Abbreviations: COP1, constitutive photomorphogenesis protein 1; TM-AFM, tapping mode-atomic force microscopy.