K19 Cys-free+His_pET-28a(+)
(Plasmid #255039)

• Purpose: Recombinant tau construct for seeding activity assays and other applications.
• Depositing Lab: Byron Caughey
• Publication: Saijo et al Acta Neuropathol. 2020 Jan;139(1):63-77. doi: 10.1007/s00401-019-02080-2. Epub 2019 Oct 16.

Backbone

• Vector backbone: pET-28a(+)
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: modified tau protein (K19)
• Species: H. sapiens (human)
• Mutation: A 3R K19 construct with a point mutation at residue 322 cysteine to serine called K19 cysteine-free (K19CF).
• Entrez Gene: MAPT (a.k.a. DDPAC, FTD1, FTDP-17, MAPTL, MSTD, MTBT1, MTBT2, PPND, PPP1R103, TAU, Tau-PHF6, tau-40)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Unknown (unknown if destroyed)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

We used a 3R K19 construct with a point mutation at residue 322 cysteine to serine called K19 cysteine-free (K19CF). This mutation prevented inter-molecular disulfide bond formation. A methionine residue was added to N-terminal residue 244 (using the numbering for full-length human tau isoform htau40), and a stop codon was added at C-terminal residue 372. The K19CF encoding sequence was designed between two restriction endonuclease enzyme sites Ndel at the 5' and Xhol at the 3' end in a bacterial expression vector pET-28a; consequently, a poly-histidine tag was expressed at the N terminus yielding K19CFh. Mutated expression cassettes were synthesized and cloned into the bacterial expression vector by GenScript.