PurposeIPTG-inducible expression of p50-ligase fusion for protein purification
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||87742||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerKitagawa et al. (PMID:16769691)
Vector typeBacterial Expression
Growth in Bacteria
Gene/Insert nameT4 DNA ligase
Entrez Gene30 (a.k.a. T4p202, lig)
- Promoter T5-lac
/ Fusion Proteins
- 6xHis (N terminal on backbone)
- human p50/NF-kB (amino acids 40-366) (N terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site SfiI (not destroyed)
- 3′ cloning site SfiI (not destroyed)
- 5′ sequencing primer pCA24N.for (GATAACAATTTCACACAGAATTCATTAAAGAG)
- 3′ sequencing primer pCA24N.rev2 — 5'-CAAATCCAGATGGAGTTCTGAGG-3' (Common Sequencing Primers)
A portion of this plasmid was derived from a plasmid made byPreviously, we PCR amplified the lig gene from pRBL (Ren et al., 1997; PMID: 9305776). A DNA fragment encoding amino acids 40-366 of human NF-kB (i.e. p50; GenBank accession number NP_003989) was amplified from plasmid pRES112 (Patrick and Blackburn, 2005 - PMID: 16008567).
Terms and Licenses
- Not Available to Industry
For complete cloning information, please see the paper's supplement.
For expression, E. coli DH5a-E cells harbouring each expression vector were grown at 37°C in LB broth containing chloramphenicol (34 µg.ml-1), to OD600 ~ 0.6. IPTG (0.4 mM, final concentration) was added as an inducer, and protein over-expression was at 28°C for 16-18 h.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pCA24N-p50-ligase was a gift from Wayne Patrick (Addgene plasmid # 87742 ; http://n2t.net/addgene:87742 ; RRID:Addgene_87742)
For your References section:Engineered DNA ligases with improved activities in vitro. Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I, Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM. Protein Eng Des Sel. 2013 Jul;26(7):471-8. doi: 10.1093/protein/gzt024. Epub 2013 Jun 10. 10.1093/protein/gzt024 PubMed 23754529