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, IICB(Glc) spans the membrane eight times. Genes encoding the circularly permuted precursors U(Cdelta)-IIA(Glc)-U(Ndelta) and U(Cdelta)-IICB(Glc)-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 IICB(Glc) 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 degrees C; 1.52 vs. 1.05 M).
Siebold
Christian
C
Departement für Chemie und Biochemie, Universität Bern, Bern, Switzerland.
Erni
Bernhard
B
D003500:Cyclization; D004926:Escherichia coli / Q000737:chemistry; D019791:Guanidine / Q000494:pharmacology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D019154:Protein Splicing; D021122:Protein Subunits; D011938:Rec A Recombinases / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D021122:Protein Subunits; D011938:Rec A Recombinases; D019791:Guanidine