Does anyone know if a streptavidin agarose column will have any affinity for lipoic acid or more specifically, lipoylated proteins? We have a protein that is lipoylated for an assay and being able to separate unlipoylated proetin would help.
I have never used a streptavidin column and I am unsure of the specific interaction with biotin. I plan to look at the crystal structure but I thought I would ask here first.
I also found a paper that gives the Ka of a synthetic lipid with a lipoic acid head group as 7 x 10^7 1/M. It seems to me that there will be some interaction with lipoic acid.
http://ac.els-cdn.com/004060909290402W/1-s2.0-004060909290402W-main.pdf?_tid=3b4be494-9b51-11e4-8b18-00000aacb35e&acdnat=1421173571_0239935cb2a96da443651facb169f2fb
What are your thoughts?
Streptavidin affinity chromatography utilizes a streptavidin-agarose matrix.Strep-tag participates in a reversible interaction it can be applied for the efficient purification of corresponding fusion proteins on affinity columns with immobilized streptavidin. The Strep-tag constitutes a nine amino acid-peptide that binds specifically to streptavidin and occupies the same pocket where biotin is normally complexed. Since the Strep-tag participates in a reversible interaction it can be applied for the efficient purification of corresponding fusion proteins on affinity columns with immobilized streptavidin. Elution of the bound recombinant protein can be effected under mild buffer conditions by competition with biotin or a suitable derivative.
Streptavidin is highly specific of Biotin. It can bind dethiobiotin (the unsulfur form of biotin) but in any case lipoic acid
Yes, lipoic acid binds streptavidin. It can be used to competitively displace Strep-tag fusion proteins from streptavidin resins (Schmidt, T. G. M. & Skerra, A. (1993). The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Prot. Engineering 6, 109-122, see note added in proof.). It binds also the closely related avidin and its affinity for avidin is 0.7 µM (Green, N. M. (1975). Avidin. Adv. Protein Chem. 29, 85-133.).
Hi Ravi, actually as a reply to Thomas comment: Is an affinity of around 1 µM really good enough for purifying lipoylated proteins? I have my doubts. I have used avidin and streptavidin columns many times in the past and never found any traces of lipoylated proteins in the purified fractions.
The unlipoylated protein will carry one extra positive charge on the lysine residue and this slight difference may be sufficient for separation. For merely analytical purposes you may follow the scheme given in this thesis; (http://theses.gla.ac.uk/139/1/Imunological_and_Biosynthetic_Studies_of_the_Human_Pyruv_%281%29.pdf). Here, lipoylated proteins are reacted with a large moelcule that shifts their molecular weight, making the lipoylated and apo forms seperable by gel electrophoresis. As indicated here (http://www.jbc.org/content/275/18/13645.long), it is also possible to directly separate apo- and holodomains by native gel electrophoresis.
Best wishes Jürgen
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