Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations

Abstract

Employing selenocysteine-containing protein fragments to form the amide bond between respective protein fragments significantly extends the current capabilities of the widely used protein engineering method, expressed protein ligation. Selenocysteine-mediated ligation is noteworthy for its high yield and efficiency. However, it has so far been restricted to solid-phase synthesized seleno-peptides and thus constrained by where the selenocysteine can be positioned. Here we employ heterologously expressed seleno-fragments to overcome the placement and size restrictions in selenocysteine-mediated chemical ligation. Following ligation, the selenocysteine can be deselenized into an alanine or serine, resulting in nonselenoproteins. This greatly extends the flexibility in selecting the conjugation site in expressed protein ligations with no influence on native cysteines. Furthermore, the selenocysteine can be used to selectively introduce site-specific protein modifications. Therefore, selenocysteine-mediated expressed protein ligation simplifies incorporation of post-translational modifications into the protein scaffold.

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 (SelM^NT) and seleno (SelM^CT) 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-SelM^CT; lane 2: same as lane 1 but after TEV protease cleavage; lane 3: MBP-SelM^NT-VMA; lane 4: MBP-SelM^NT thioester following cleavage of MBP-SelM^NT-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. SelM^NT 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.

Figure 2

ESL can be used to generate non-selenoproteins and to introduce a unique site for labeling. Panels a-d) Chemical ligation of E. coli Trx with Sec at position 94 and subsequent deselenization to form wild-type Trx. (a) The structure of Trx A94U based on PDB entry 2TRX. Red: thioester fragment; Green: seleno-fragment; Yellow: sulfur atoms; Orange: selenium atom. (b) The design of thioester (Trx^NT) and seleno (Trx^CT) fragments. (c) Deconvoluted ESI-MS of Trx A94U. (d) Deconvoluted ESI-MS of wild-type Trx generated by deselenization of Sec to Ala (the * denotes Trx A94S). (e) The enzymatic activity of Trx generated by deselenization and that of Trx prepared by heterologous expression are similar as measured by insulin turbidity assays. Data represent mean±s.d. (n=3). (f) Labelling of Trx A94U with a thiophosphate (the peak marked with ** is a contamination from the commercial thiophosphate). (g) Conjugation of Trx A94U to ubiquitin G76C via the formation of a selenylsulfide bond. (h) Conjugation of Trx A94U to ubiquitin G76U via the formation of a diselenide bond. Panels i-j) Due to Sec’s low pK_a it can be selectively alkylated. (i) A mixture of ubiquitin G76C and ubiquitin G76U prior to alkylation. (j) The same sample from panel i alkylated with MM(PEG)₂₄. Only ubiquitin G76U was alkylated.

Figure 3

Chemoselective dehydroalanine formation from Sec. (a) Sec deselenization into dehydroalanine can be promoted through two successive alkylation steps. The reaction can be carried out selectively, even when cysteines are present, due to difference between Sec and Cys pK_as. (b) Dehydroalanine formation in SelM at pH 6.5 showing complete conversion. (c) The sample from panel b treated with excess iodoacetamide at pH 7.0. The sole Cys was available for alkylation at that pH even though it was in proximity to the dehydroalanine site. (d) Tandem mass of trypsin-digested sample from panel c further confirms the presence of dehydroalanine in position 48. Red color indicates ions containing dehydroalanine. The purple star denotes alkylation by iodoacetamide.