We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure–function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.
We thank all members of our laboratories for helpful discussions. E.A.L., C.K., P.F.S., M.W., and S.B. acknowledge funding from the BW Stiftung. E.A.L. acknowledges additional support from the Emmy Noether program. E.A.L. and C.S. are grateful for funding by SPP1623 of the Deutsche Forschungsgemeinschaft. I.B. is funded by the European Commission Framework Programme 7 (FP7) ComplexINC project (contract no. 279039). P.S.-B. acknowledges funding from the Laura Bassi Centres of Expertise initiative for the Centre of Optimized Structural Studies, project 253275. M.W. thanks the KSOP for financial support. P.S.T. is supported by the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND. The Wellcome Trust generously funded this work through a Senior Research Fellowship to J.R. (103139), a Centre core grant (092076), and an instrument grant (108504). We also thank the members of the EMBL Genomics Core Facility for sample processing and sequencing, as well as the EMBL FACS facility for technical support.
Integrated supplementary information
List of quantified cross-linked peptides between TAF11 and TAF1334→DiAzKs.