The position of the heterologous domain can influence the solubility and proteolysis of beta-galactosidase fusion proteins in E. coli.
The VP1 protein (23 kDa) of the foot-and-mouth disease virus has been produced in MC1061 and BL21 E. coli strains as beta-galactosidase fusion proteins, joined to either the amino and/or the carboxy termini of the bacterial enzyme. In BL21, devoid of La protease, all the recombinant fusion proteins are produced at higher yields than in MC1061, and occur mainly as inclusion bodies. The fusion of VP1 at the carboxy terminus yields a protease-sensitive protein whose degradation releases a stable, enzymatically active polypeptide indistinguishable from the native beta-galactosidase. On the contrary, when the same viral domain is fused to the amino terminus, the resulting chimeric protein is resistant to proteolysis even in the soluble form. These data demonstrate that the position of the heterologous domain in beta-galactosidase fusion proteins would not be irrelevant since it can dramatically influence properties of biotechnological interest such as solubility and proteolytic resistance.
Corchero
J L
JL
Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Viaplana
E
E
Benito
A
A
Villaverde
A
A
D005537:Aphthovirus / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D036022:Capsid Proteins; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D002479:Inclusion Bodies / Q000378:metabolism; D007763:Lac Operon; D015394:Molecular Structure; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D012995:Solubility; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*
D036022:Capsid Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases