Selenocysteine (Sec; U in one-letter code), the 21st amino acid existing in all selenoproteins, has unique biochemical properties due to its selenium atom, including a low pK a and a high reactivity with many electrophilic agents. Mammalian thioredoxin reductase (TrxR) is a selenoprotein with a redoxactive selenolthiol motif within its C-terminal tetrapeptide, -Gly-Cys-Sec-Gly-COOH (GCUG). We have used that motif as a "Sel-tag" for recombinant proteins produced in Escherichia coli and shown that it could be exploited for selenolate-targeted one-step purification as well as residue-specific fluorescent labeling with 4(5)-(iodoacetamido)fluorescein (5-IAF) and radiolabeling with either gamma emitters ( 75 Se) or positron-emitting radionuclides ( 11 C) (1).
We here extend these studies to other C-terminal tetrapeptide motifs, suggested by our prior results using mutants of Drosophila melanogaster TrxR. This enzyme has, instead of selenocysteine, a cysteine residue which is activated by flanking serine residues (2). In order to compare the reactivity of this type of tetrapeptide with our previous GCUG tag, we here produced recombinant Fel d 1, an allergen from the domestic cat (Felis domesticus), with two dithiol (-GCCG and -SCCS) and two selenolthiol (-GCUG and -SCUS) C-terminal tags. We subsequently assessed the use of these tags in one-step purification, fluorescent labeling and positron emitter labeling.
Phenylarsine oxide (PAO) columns could be utilized to efficiently purify all four Fel d 1 variants from whole bacterial lysates in a single purification step. The protein obtained was apparently homogeneous, as judged by Coomassie-stained SDS-PAGE analysis. Yields were about 5-10 mg pure protein from 1 liter bacterial culture.
We next found that all four tags could be fluorescent labeled with 5-IAF, with -GCCG being the least reactive and -SCUS being the most easy to label. Finally, we assessed the use of both the selenolthiol and dithiol tags for residue-selective labeling with a commonly used positron-emitting electrophilic precursor, [ 11 C]CH 3 I. Similar to the fluorescent labeling, both the selenolthiol and dithiol variants could be radiolabeled with 11 C by incubating with reduced protein at room temperature for 10-25 min. The labeling efficiency of the selenolthiol motifs were, however, about 2-3-fold higher than the dithiol variants.
In summary, these results illustrate that not only the Sel-tag but also derivatives thereof can be used for purification and residue-specific radiolabeling. The differences observed in the labeling efficiencies of the tags merit further study into the mechanisms behind their reactivities. The fact that this approach permits efficient labeling of proteins with even such a short-lived tracer as [ 11 C]CH 3 I is very promising for its use in developing novel applications for proteins labeled with a range of positronemitting nuclides.