A New Tool for Custom Protein Design and Engineering - DH10 Bac-TAG

Genetic code expansion is emerging as a major tool for protein engineering. It allows for introduction of non canonical amino acids with various functional groups for e.g. labeling of proteins for single molecule studies or super resolution microscopy, cross-linking of proteins or attaching a post-translational modification of choice. This system is well established in E. coli, which permits good expression yields of recombinant proteins. However, such simple laboratory hosts are not well suited for expression of large multi-protein complexes with ideally native eukaryotic posttranslational protein modifications. To achieve this, one of the most widely used systems is the MultiBac system, which is a versatile platform to easily generate large protein assemblies and express them in the eukaryote Spodoptera frugiperda (Sf21).Here we present a strategy how to perform genetic code expansion in Sf21 cells, combining the benefits of advanced protein engineering with the power of the MultiBac system into a single, versatile expression platform.We shuffled the orthogonal synthetase-tRNA pair PylRS/tRNAPyl from Methanosarcina mazei into a commonly used Bacmid, resulting in a new DH10 Bac-TAG cell line. The system permitted high level expression of proteins at low cost. We further demonstrate the system for expression of multi-protein assemblies, fluorescence labelling and glyco engineering for custom biologics.