Insertion of a 27 amino acid viral peptide in different zones of Escherichia coli beta-galactosidase: effects on the enzyme activity.
Seven internal, putatively exposed regions of Escherichia coli beta-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides. Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique BamHI restriction sites. By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme. Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities. The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate to incorporate and study biological properties of heterologous peptides in correctly folded beta-galactosidase chimeric proteins.
Benito
A
A
Institut de Biologia Fondamental, Universitat Autònoma de Barcelona, Spain.
Villaverde
A
A
D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D036022:Capsid Proteins; D015342:DNA Probes; D001616:beta-Galactosidase