Synthesis and Purification of Azide-Tagged IFN-γ

We synthesized azide-tagged IFN-γ (azIFN-γ) as previously reported,^{9 (link)} with no deviations. Briefly, azIFN-7 was inserted into a pET28a vector (GenScript), while N-myristoyltransferase was inserted into a pET11c vector (generously provided by Dr. Edward Tate, Imperial College London²⁴). We co-expressed both proteins in BL21 (DE3) E. coli, using 12-azidododecanoic acid (synthesized as in^{9 (link)}) as the azide tag, isolated the protein under denaturing conditions via nickel-nitrilotriacetic acid (Thermo Scientific), refolded it, and performed the final purification using fast protein liquid chromatography (FPLC, GE AKTApurifier 10 with Frac-950 collector - buffers in,^{9 (link)} procedure in^{25 (link)}). We determined concentration for all experiments using the bicinchoninic acid (Micro BCA, Thermo Scientific) assay.

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Ham T.R., Cox D.G, & Leipzig N.D. (2019). Concurrent delivery of soluble and immobilized proteins to recruit and differentiate neural stem cells. Biomacromolecules, 20(9), 3445-3452.

Publication 2019

pET28a vector nickel-nitrilotriacetic acid AKTApurifier Frac-950 collector Micro BCA

12 azidododecanoic acid Assay Azide Bicinchoninic acid Buffers E coli Ifn γ Liquid chromatography N myristoyltransferase Nickel nitrilotriacetic acid Protein Vector

Corresponding Organization : University of Akron

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Variable analysis

independent variables

Synthesis of azide-tagged IFN-γ (azIFN-γ)
Co-expression of azIFN-γ and N-myristoyltransferase in BL21 (DE3) E. coli
Use of 12-azidododecanoic acid as the azide tag

dependent variables

Protein isolation under denaturing conditions via nickel-nitrilotriacetic acid
Protein refolding
Final protein purification using fast protein liquid chromatography (FPLC)
Protein concentration determination using bicinchoninic acid (Micro BCA) assay

control variables

No deviations from previously reported procedures for the synthesis of azIFN-γ

controls

Positive control: Previously reported procedures for the synthesis of azIFN-γ
Negative control: Not explicitly mentioned

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