Advanced expression: Employing SpyTag & SpyCatcher for post-translational fusion
At a certain point it will be necessary to think about transgenic moss which is able to degrade several different xenobiotics using different effector molecules. In order to prepare for these posterior needs we decided to integrate the SpyCatcher and SpyTag system into our project [Ref]. This Spy-system allows for the creation of posttranslational protein fusion based on a covalent bond which is formed between the side chains of residues of a SypCatcher and a SpyTag.
Therefore it is possible to create a protein fusion of the SypTag with a recombinant effector protein which is expressed separately (e.g. with anothe expression strength) and becomes in the secretory pathway fused to a receptor which contains the SypCatcher. By these means it becomes possible to express the SERK-receptor under a strong promoter and to adjust the expression of different effector proteins to their particuluar necessity (Fig. x B).
On the other hand enzymatic effectors might be active as multimeric proteins and thus not every subunit can be fused to a receptor for steric reasons. In this case the application of the SpyTag system also seems advantageous as it allows the multimeric protein to assemble into its functional form before it becomes immobilized to the outer side of the cellular membrane by its SpyTags (Fig. x C).