Expression of functional beta-galactosidase containing the coxsackievirus 3C protease as an internal fusion.

Alpha complementation of beta-galactosidase (beta gal) is intracistronic and requires interaction between the alpha donor region (residues 3-41) and alpha acceptor fragment (produced by M15). We have constructed two plasmids which direct the synthesis of hybrid beta gal: coxsackievirus proteins in Escherichia coli. One plasmid, pBD1045, encodes an enzymatically active 3C protease of coxsackievirus B3 fused between the amino-terminal 79 amino acids of beta gal (containing the alpha donor region) and amino acids 80 to 1023 (alpha acceptor region). A second plasmid, pBD1043 encodes an inactive 3C protease and results in a fusion of 260 coxsackievirus amino acids between residues 79 and 80 of the beta gal monomer. Both hybrid proteins expressed by these constructs have beta-galactosidase activity regardless of whether the viral protease (183 amino acids) is autocatalytically cleaved out of the chimeric protein (pBD1045) or remains as part of a fusion protein (pBD1043). The implications of these results for structural flexibility of the complemented beta-galactosidase enzyme are discussed.