Applications of intein mediated ligation

Abstract

Intein mediated ligation provides a site-specific method for the attachment of a wide range of molecular probes to proteins.  The method has some inherent practical difficulties which have limited its application.  The use of this valuable technique for the site specific fluorescent labelling and controlled electrode immobilisation of proteins was investigated, with intein mediated ligation chosen as a method due to its site specificity.  Plasmids for the expression of target-intein fusion proteins were assembled and fusion protein expression optimised in a range of six E. coli strains, four target-intein fusion proteins were purified by Ni affinity chromatography.  The reaction of a model target-intein fusion protein with a fluorescent cysteine derivative was investigated under a range of conditions including anaerobicity of the reaction and the addition of low molecular weight thiols.  It was found that the ligation of the fluorophore to the target protein is critically dependant upon the degree of oxidation of the fluorescent cysteine label.  Unusually it was shown that efficient direct reaction of the fluorescent label with the target-intein thioester was possible, but only under rigorously anaerobic conditions.  Optimal, more reliable conditions for efficient reaction include anaerobic reaction with the addition of thiophenol (10 mM) or aerobic reaction with the further addition of the water soluble reducing agent, tricarboxyethylphosphine.

The glassy carbon electrode surface was oxidatively modified with diaminodecane, which was coupled with a protected cysteine derivative.  Each electrode derivatization step was characterised electrochemically and by XPS.  Cysteine deprotection was followed by reaction with BioB-intein, although protein was present at the electrode surface (as observed by XPS); it was unclear whether the protein has been attached via intein mediated ligation or had non-specifically adsorbed.

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