Streamlined construction of robust heteroprotein complexes by self-induced in-cell disulfide pairing.

Biomolecules and their functional subdomains are essential building blocks in the creation of multifunctional nanocomplexes. Methyl-binding domain protein 2 (MBD2) and p66α stand out as small α-helical motifs with an ability to self-assemble into a heterodimeric coiled-coil, making them promising building units. Yet, their practical use is hindered by rapid dissociation upon dilution. In this study, novel fusion tags, MBD2 and p66α variants, were developed to covalently link during co-expression in E. coli SHuffle. Through strategic placement of cysteine at each α-helix's terminus, intracellular crosslinking occurred with high specificity and yield, facilitated by preserved α-helical interactions. This instant disulfide bonding in the oxidative cytoplasm of E. coli SHuffle efficiently overcame the need for inefficient in vitro oxidation and protein extraction prone to creating non-specific adducts and suboptimal bioprocesses. In contrast to their wild-type counterparts, the GFP-mCherry protein complex cross-linked by the fusion tags maintained the heterodimeric state even under extensive dilution. The fusion tags, when combined with the E. coli SHuffle system, allowed for the streamlined co-expression of a stable protein complex through self-induced intracellular cysteine coupling. The approach demonstrated herein holds great promise for producing multifunctional and robust heteroprotein complexes. • A pair of Cys-mutated α-helices function as fusion tags for stable protein dimerization • Tagged proteins are self-assembled and crosslinked intracellularly with high yield. • Crosslinking is driven by cognate α-helical interaction and disulfide-bond formation. • The oxidative bacterial co-expression system facilitates in-cell disulfide pairing. • Fusion tags enable the formation of a heteroprotein complex with improved stability. [ABSTRACT FROM AUTHOR]