Genetic code expansion for multiprotein complex engineering

• Published in: Nature methods Vol. 13; no. 12; pp. 997 - 1000
• Main Authors: Koehler, Christine, Sauter, Paul F, Wawryszyn, Mirella, Girona, Gemma Estrada, Gupta, Kapil, Landry, Jonathan J M, Fritz, Markus Hsi-Yang, Radic, Ksenija, Hoffmann, Jan-Erik, Chen, Zhuo A, Zou, Juan, Tan, Piau Siong, Galik, Bence, Junttila, Sini, Stolt-Bergner, Peggy, Pruneri, Giancarlo, Gyenesei, Attila, Schultz, Carsten, Biskup, Moritz Bosse, Besir, Hueseyin, Benes, Vladimir, Rappsilber, Juri, Jechlinger, Martin, Korbel, Jan O, Berger, Imre, Braese, Stefan, Lemke, Edward A
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.12.2016; Nature Publishing Group
• Subjects: 101/58; 13/44; 14/33; 14/34; 631/1647/1511; 631/1647/338/469; 631/45/612; 631/92/56; 631/92/96; 82/1; Animals; Baculoviridae - genetics; Baculoviridae - metabolism; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical Engineering/Biotechnology; brief-communication; Cell Culture Techniques; Energy transfer; Engineering; Fluorescence Resonance Energy Transfer - methods; Genes; Genetic aspects; Genetic Code; Genetic engineering; Genetic Vectors; Genomes; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Humans; Life Sciences; Molecular biology; Multiprotein Complexes - biosynthesis; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; Organisms; Plasmids; Protein Engineering - methods; Proteins; Proteomics; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Sf9 Cells; Spodoptera; Transfer RNA; Viral Proteins - biosynthesis; Viral Proteins - chemistry; Viral Proteins - genetics; Yeast; Germany; United Kingdom > UK
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.4032

A method for producing multiprotein complexes engineered with site–specifically introduced noncanonical amino acids is described, enabling applications in biochemical and biophysical analysis, as well as in biotechnology. 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.