Intein-mediated cyclization of a soluble and a membrane protein in vivo: function and stability Original Research Article

Cyclized subunits of the E. coli glucose transporter were produced in vivo by intein mediated trans-splicing. IIAGlc is a β-sandwich protein, IICBGlc spans the membrane eight times. Genes encoding the circularly permuted precursors UCΔ-IIAGlc-UNΔ and UCΔ-IICBGlc-UNΔ were assembled from DNA fragments encoding the 3′ and 5′ segments of the recA intein of M. tuberculosis and crr and ptsG of E. coli, respectively. A 20-residues long, Ala-Pro rich linker peptide and/or a histidine tag were used to join the native N- and C-termini in the cyclized proteins. The cyclized proteins complemented growth of glucose auxotrophic strains. Purified, cyclized IIAGlc and IICBGlc had 100 and 25%, respectively, of wild-type glucose phosphotransferase activity. They had an increased electrophoretic mobility, which decreased upon linearization of the proteins with chymotrypsin. Cyclized IIAGlc displayed increased stability against temperature and GuHCl-induced unfolding (75 vs. 70 °C; 1.52 vs. 1.05 M).