PurposeExpresses human IL-2 (C125S)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||104348||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4751
- Total vector size (bp) 5115
Vector typeBacterial Expression
Growth in Bacteria
Growth instructionsBL21 for protein expression
Copy numberHigh Copy
Gene/Insert nameHuman IL-2 (C125S)
SpeciesH. sapiens (human)
Insert Size (bp)410
MutationC125S mutation (TGT -> TCC)
Entrez GeneIL2 (a.k.a. IL-2, TCGF, lymphokine)
- Promoter T5
/ Fusion Protein
- MRGSHHHHHGS-tag (N terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site BamHI (not destroyed)
- 3′ cloning site HindIII (not destroyed)
- 5′ sequencing primer CGGATAACAATTTCACACAG
- 3′ sequencing primer GTTCTGAGGTCATTACTGG (Common Sequencing Primers)
Like the FDA approved Proleukin (Aldesleukin), the sequence contains a C125S mutation (TGT -> TCC) to prevent cysteine mispairing in E. coli, but this does not affect biological activity (Wang et al. 1984) Expression and inclusion body preparation was carried out essentially as described in Carmenate et al. (2013), except that the 6M guandine solubilised IL-2 was applied directly to an NTA column at 1 column volume / minute. LPS was removed with 0.1% Triton X114 / TE washes following by on-column re-folding with TE buffer. NTA-purified His-IL-2 exhibited identical activity to commercially sourced (tag-free) IL-2 (Peprotech).
Wang A, Lu SD, and Mark DF. Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science 1984; 224:1431-1433.
Carmenate T, Pacios A, Enamorado M, Moreno E, Garcia-Martinez K, Fuente D, and Leon K. Human IL-2 mutein with higher antitumor efficacy than wild type IL-2. J. Immunol. 2013; 190:6230-6238.
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:pQE80L-IL2 C125S was a gift from Alexander McLellan (Addgene plasmid # 104348 ; http://n2t.net/addgene:104348 ; RRID:Addgene_104348)