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

Abstract

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).

• Authors: Christian Siebold, Bernhard Erni
• Journal: Biophysical chemistry (Biophys Chem) Vol. 96 Issue 2-3 Pg. 163-71 (May 02 2002) ISSN: 0301-4622 [Print] Netherlands
• PMID: 12034438 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Membrane Proteins
• Monosaccharide Transport Proteins
• Protein Subunits
• Rec A Recombinases
• Guanidine

Topics

• Cyclization
• Escherichia coli (chemistry)
• Guanidine (pharmacology)
• Membrane Proteins (biosynthesis, chemistry)
• Monosaccharide Transport Proteins (biosynthesis, chemistry)
• Protein Conformation
• Protein Denaturation (drug effects)
• Protein Splicing
• Protein Subunits
• Rec A Recombinases (metabolism)
• Solubility
• Structure-Activity Relationship