Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation...

Ice-nucleation protein (INP), an outer membrane protein from Pseudomonas syringae, is able to catalyze the ice crystal formation of supercooled water. It was exploited for anchoring of Bacillus subtilis carboxymethylcellulase (CMCase) on the surface of Escherichia coli. A surface anchoring vector, pGINP21M, was created that contains the multicloning sites including BamHI, SmaI and EcoRI at the end of the 3' flanking region encoding the C-terminus of INP instead of the stop codon for subcloning the foreign genes. The CMCase gene was in-frame subcloned for making INP-CMCase fusion proteins. The ability of this vector for directing the actual synthesis of INP-CMCase fusion proteins was confirmed by Western blotting analysis. CMCase targeted on the surface of cells was verified by measuring whole cell CMCase activity and ice-nucleation activity. CMCase activity was mainly detected on the cell surface whereas no enzyme activity was detected in the culture supernatant. Ice-nucleation activity was also maintained even if an INP-CMCase hybrid was made. This means that the fusion protein is functionally expressed and has its biological conformation on the surface. INP-CMCase fusion proteins were stable in the stationary phase. INP deleted of the repeating domain, thus producing no ice-nucleation activity, could also direct CMCase on the cell surface. This suggests that it has the secretion and targeting signal to the outer membrane.