Tris is a non-innocent buffer during intein-mediated protein cleavage

Area of Science:

• Biochemistry
• Protein Chemistry
• Analytical Chemistry

Background:

• Self-cleavable protein splicing elements offer tag-free recombinant protein purification.
• A key challenge is the formation of reactive thioester intermediates during splicing.
• These intermediates can lead to undesired side reactions and adduct formation.

Purpose of the Study:

• To investigate adduct formation during self-cleavable protein purification.
• To evaluate the impact of buffer components on thioester intermediate stability.
• To explore potential applications of the self-splicing system for protein modification.

Main Methods:

• Electrospray ionization mass spectrometry (ESI-MS) was used to monitor adduct formation.
• The purification system was tested using Triticum aestivum (wheat) E(c)-1 metallothionein.
• Reactions were analyzed in the presence of various buffer components, including tris(hydroxymethyl)aminomethane (Tris).

Main Results:

• Tris buffer was identified as a non-innocent component, reacting with thioester intermediates to form a stable, previously unreported adduct.
• The study demonstrated that Tris buffer must be used cautiously with this purification system.
• High levels of N-formylmethionine were observed when the protein was expressed in cadmium-supplemented media.

Conclusions:

• Tris buffer can interfere with self-cleavable protein purification by forming adducts with reactive intermediates.
• The findings necessitate careful buffer selection for efficient and clean purification.
• The self-splicing strategy holds potential for direct C-terminal protein or peptide modification.