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The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB(Glc) subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB(Glc) are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB(Glc) and IIA(Glc) were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA(Glc) and IICB(Glc) had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding.

Original publication

DOI

10.1016/s0014-5793(01)02705-3

Type

Conference paper

Publication Date

08/2001

Volume

504

Pages

104 - 111

Addresses

Departement für Chemie und Biochemie, Universität Bern, CH-3012, Bern, Switzerland

Keywords

Escherichia coli, Phosphoenolpyruvate, Carbohydrates, Monosaccharide Transport Proteins, Recombinant Fusion Proteins, RNA, Messenger, Temperature, Transcription, Genetic, Amino Acid Sequence, Base Sequence, Protein Conformation, Protein Structure, Tertiary, Protein Folding, Biological Transport, Phosphorylation, Dose-Response Relationship, Drug, Mutation, Models, Biological, Models, Molecular, Time Factors, Molecular Sequence Data, Carbohydrate Metabolism