An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation
protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer
membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step
the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological
support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which
allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised
proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our
results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic
anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the b-glycoside hydrolase (SsbGly)
from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the
improvement in the utilisation of biocatalysts of biotechnological interest.