Surface Anchoring on Lactococcus lactis by Covalent Isopeptide Bond

Abstract

Display of recombinant proteins on the bacterial surface is an emerging research area with wide range of potential biotechnological applications. Because of its GRAS (generally recognized as safe) status, lactic acid bacterium Lactococcus lactis represents an attractive host for surface display and promising vector for in situ delivery of bioactive proteins. The present study focused on finding a new alternative approach for surface display on Lactoccocus lactis. We developed a system that enables the formation of irreversible isopeptide bonds on the surface of Lactococus lactis. This was achieved through the following two protein/peptide pairs, SpyCatcher/SpyTag and SnoopCatcher/SnoopTag. 1-3 Attachment of tagged model protein B domain to the cell surface of Lactococccus lactis displaying the corresponding catcher protein was demonstrated using flow cytometry. We demonstrated effective use of aforementioned protein anchors which thus represent a promising alternative to established approaches for surface display on Lactoccocus lactis.

Surface Display of Complex Enzymes by in Situ SpyCatcher‐SpyTag Interaction

“…SC and ST form a covalent isopeptide bond between a proximal aspartic acid and lysine residue, thereby enabling the covalent coupling of correctly folded proteins that are independently expressed as genetic fusions with the SC and ST domains. The SC‐ST coupling occurs over a wide range of physiological conditions, does not require reagents and has therefore also been applied for in vivo applications inside bacterial cells …”

“…The SC-ST coupling occurs over a wide range of physiological conditions, does not require reagents and has therefore also been applied for in vivo applications inside bacterial cells. [21][22][23][24][25][26] To extend the toolbox for surface display of complex, high molecular weight enzymes on E. coli cells, we here report on the investigation of a novel modular surface display method in which the membrane anchor and the passenger are expressed separately and assembled by post-translational bioconjugation via SC-ST coupling (Figure 1). As this allows independent folding of the anchor and passenger proteins, we reasoned that our approach might reduce unfavourable domain interactions and misfolding, thereby increasing the display efficiency and the whole-cell biocatalytic activity.…”

Safety Aspects of Genetically Modified Lactic Acid Bacteria

“…An alternative approach for covalent heterologous surface display on L. lactis was achieved through formation of isopeptide bonds between the SpyCatcher/SpyTag and SnoopCatcher/SnoopTag protein/peptide pairs. The tagged model protein-B domain was successfully attached to the cell surface of L. lactis to display the corresponding catcher protein [123].…”