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

Cyclized subunits of the E. coli glucose transporter were produced in vivo by intein mediated trans-splicing. IIA(Glc) is a beta-sandwich protein, IICBGlc spans the membrane eight times. Genes encoding the circularly permuted precursors U-CDelta-IIA(Glc)-U-NDelta and U-CDelta-IICBGlc-U-NDelta 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 IIA(Glc) 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 IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding (75 vs. 70 degreesC; 1.52 vs. 1.05 M). (C) 2002 Elsevier Science B.V. All rights reserved.