pKK-TEV-ProteinA
(Plasmid #105788)

• Purpose: (Empty Backbone) Expression of your protein of interest in fusion with two IgG binding domains of Staphylococcus aureus protein A at the C-terminus (cleavable by TEV) (PMID: 3507693, 10504710). Protein purification.
• Depositing Lab: Andrzej Dziembowski
• Publication: Szczesny et al PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018.

Backbone

• Vector backbone: pcDNA5/FRT/TO
• Backbone manufacturer: Invitrogen
• Vector type: Mammalian Expression ; Flp-In competent
• Selectable markers: Hygromycin
• Tag / Fusion Protein:
  • TEV-ProteinA (C terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: None
• Tag / Fusion Protein:
  • TEV-ProteinA (C terminal on backbone)

Resource Information

• Supplemental Documents:
  • pKK-TEV-ProteinA.txt

Terms and Licenses

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

Depositor Comments

This vector is a member of the pKK vector family. Its cloning site was optimized for the SLIC approach, however, a traditional restriction and ligation based approach is also possible (see Szczesny RJ et al. for detailed protocols; bioRxiv, doi: https://doi.org/10.1101/160101) This vector is a derivate of the pcDNA5/FRT/TO plasmid (Invitrogen) and is suitable for stable cell line generation using the Flp-In system. Transcription of the gene of interest is driven by a tetracycline responsive CMV promoter. A given coding sequence can be cloned into all pKK vectors using just three universal PCR primers and a ligation-independent DNA cloning method. Protein A of Staphylococcus aureus binds to IgG of different origin which can be used for affinity purification or antibody-based detection. Protein A (i.e. two IgG-binding domains of protein A) is often used for tandem affinity purification, in which a protein of interest is expressed in fusion with two different tags (for example Protein A-calmodulin binding peptide) (PMID: 3507693, 10504710). In this vector only Protein A is encoded.