title: Reversible activation of a cryptic cleavage site within E. coli beta-galactosidase in beta-galactosidase fusion proteins.
abstract: The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.
authors.affiliation: Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Viaplana
authors.forename: J X
authors.initials: JX
authors.lastname: Feliu
authors.forename: J L
authors.initials: JL
authors.lastname: Corchero
authors.forename: A
authors.initials: A
authors.lastname: Villaverde
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D020689:Exopeptidases; D005822:Genetic Vectors; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017421:Sequence Analysis; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C081149:viral protein 60, rabbit hemorrhagic disease virus; D001616:beta-Galactosidase; D020689:Exopeptidases; D010447:Peptide Hydrolases