Reversible activation of a cryptic cleavage site within E. coliβ-galactosidase in β-galactosidase fusion proteins

• Published in: Biochimica et biophysica acta 1997-12, Vol.1343 (2), p.221-226
• Main Authors: Viaplana, Elisenda, Feliu, Jordi X, Corchero, José L, Villaverde, Antonio
• Format: Article
• Language: English
• Subjects: beta-Galactosidase - chemistry; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Blotting, Western; E. coli; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli - enzymology; Exopeptidases; Folding; Genetic Vectors; Inclusion body; Models, Molecular; Peptide Hydrolases - metabolism; Protease; Protein Conformation; Recombinant Fusion Proteins - metabolism; Recombinant protein; Sequence Analysis; Viral Structural Proteins - genetics; Viral Structural Proteins - metabolism; β-Galactosidase
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(97)00114-3

The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of β-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 β-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 β-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in β-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.