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. Author manuscript; available in PMC: 2018 Feb 23.
Published in final edited form as: J Am Chem Soc. 2017 Feb 27;139(9):3430–3437. doi: 10.1021/jacs.6b10991

Figure 1.

Figure 1

(a) Principle of expressed selenoprotein ligation (ESL). The seleno-fragment, fused with MBP, is expressed in E. coli in selenocystine enriched medium. The complementary thioester protein fragment is prepared via intein technology. TEV protease cleaves MBP from the seleno-fragment and the protein of interest (POI) is then spontaneously generated through amide bond formation. (b) Applications of Sec-mediated ligation. Panels C-H: ESL preparation of SelM. (c) Design of SelM fragments. Red: thioester fragment; Green: seleno-fragment; Yellow: sulfur atom; Orange: selenium atom. (d) The design of thioester (SelMNT) and seleno (SelMCT) fragments. SelM’s ER targeting sequence (residues 1 to 24) was omitted as it is cleaved in vivo. (e) SelM ligation at 25 °C and pH 7.0 monitored by SDS-PAGE under reducing conditions: Lane 1: MBP-SelMCT; lane 2: same as lane 1 but after TEV protease cleavage; lane 3: MBP-SelMNT-VMA; lane 4: MBP-SelMNT thioester following cleavage of MBP-SelMNT-VMA with thiols and subsequent purification; lanes 5-7 the formation of SelM monitored on days 0, 1, and 2. Lane M: molecular mass standards. SelMNT cannot be detected because of its 2 kDa mass. (f) Deconvoluted ESI-MS of intact SelM. (g) ESI-MS spectrum of the SelM peptide containing the Sec residue alkylated with iodoacetamide (purple star). (h) Tandem MS sequencing of the peptide from panel G confirms Sec’s presence in SelM. Fragment ions that contain Sec are colored red. (i) Superimposed CD spectra of SelM, before and after refolding, and SelM U48C prepared by heterologous expression.