Tris is a non-innocent buffer during intein-mediated protein cleavage.
Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.
Peroza
Estevão A
EA
Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Freisinger
Eva
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D004229:Dithiothreitol / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D047668:Inteins; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D014908:Triticum / Q000235:genetics; D014325:Tromethamine / Q000378:metabolism*
D010940:Plant Proteins; D014325:Tromethamine; D008715:Methionine; D004229:Dithiothreitol