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

Abstract

Alpha complementation of beta-galactosidase (βgal) 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 βgal: 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 βgal (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 βgal 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.