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.
Windheuser
M G
MG
Schering-Plough Research, Bloomfield, New Jersey 07003.
Dwyer
S
S
Dasmahapatra
B
B
D000086042:3C Viral Proteases; D001483:Base Sequence; D015153:Blotting, Western; D002678:Chimera; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004770:Enterovirus / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D001616:beta-Galactosidase; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases