Proteomics and Synthetic Biology

Much of the success to understand the genetic code in the past two decades has stemmed from the development of experimental methods to introduce artificial coding rules in vivo. Suppression (or read-through) of the stop codon (commonly the amber codon TAG) by the orthogonal translation apparatus enables the genetic code expansion. The key engineered orthogonal components, aminoacyl-tRNA synthetase and the suppressor tRNA (RS/tRNA) pair, can be transiently introduced into living cells via DNA injection, transformation, transfection, electroporation and viral delivery. The presence of the RS/tRNA allows the incorporation of an unnatural amino acid (Uaa) with unique properties such as light-sensitive, chemical-reactive into proteins in situ.

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Uaa incorporations in mammalian cells: We have established an efficient method to encode Uaas in mammalian HEK293 cells, by co-transfection of orthogonal components (aminoacyl-tRNA synthetase and the suppressor tRNA pairs). We have successfully encoded tyrosine analogues in GPCRs, NMDA receptors and membrane transporters.

Light sensitive NMDA receptors: We have demonstrated that protein functions can be irreversibly changed when inserting photo-cross-linking Uaa (such as AzF) at specific sites of the proteins. Uaas containing azobenzene moiety allow the reversible switching of protein functionalities.

Fluorescent labeling of proteins: We have applied the bioreactive Uaas (such as AzF and TCO*), which enables the site-specific bioorthogonal labelling complementary to the cysteine labeling method for fluorescent imaging of receptors.

Engineering aminoacyl-tRNA synthetase: We have applied computational protein design to engineer E. coli tyr-tRNA synthestase to take D-Tyr as its substrate.

Transgenic mice and zebrafish: Our recent demonstration of transgenic zebrafish and mice inheriting genetic components to decode the amber stop codon with AzF has provided valuable vertebrate model animals, which will facilitate a versatile array of applications to control protein functions in vivo.

Site-specific interactome: We will combine systemic computational tools and mass-spectrometry to discover the interactomes of membrane transporters, which are promising drug target for cancer and brain diseases. 

Highlights

  • Implemented computational protein design to create de novo membrane proteins mimicking photosynthetic proteins
  • Established genetic code expansion in GPCRs by encoding the unnatural amino acid p-azido-L-phenylalanine (AzF) as an infrared probe 
  • Discovered that rhodopsin undergoes major conformational changes already at early stages of the photo-activation process by implementing Fourier Transform Infrared spectroscopy
  • Pioneered in the genetic code expansion in Xenopus laevis oocytes
  • Engineered light-sensitive NMDA receptors with site-specific incorporated AzF and TCO*
  • Created transgenic mice and zebrafish lines with an expanded genetic code
  • Generated D-Tyr aminoacyl tRNA-synthetase by combining computational protein design and experimental characterization

Future directions

  1. To apply methods associated with the genetic code expansion to understand the structure-function relationship of proteins. The site-specific incorporation of Uaas, with chemical and spectroscopic properties absent in the natural amino acids repertoire, has been proved to be very useful to correlate the functional changes arising from a single point mutation.
  2. To investigate the possibility of building basic functional proteins with a minimal set of amino acid repertoire. This fundamental approach is driven by the interest in understanding the standard genetic code. Theoretical and bioinformatic investigations suggest that small set of amino acids in the genetic code bear the property to generate all major secondary structure motifs observed in proteins. Results in this field will also help clarifying current concepts and ideas about the origin and evolution of the genetic code.

Collaborations

  • Thomas P. Sakmar, GPCRs biology and signal transduction. Rockefeller University, USA
  • Chonggang Yuan, Neurodegenerative diseases. East China Normal University, China. 
  • Dali Li, Rodent Genomic editing and gene therapy, East China Normal University, China. 
  • Lei Wang, Genetic code expansion, University of California, San Francisco, USA. 
  • Jeff Holst, Prostate cancer and amino acid transporters, University of Sydney, Australia. 
  • Péter Kele, UAA mediated bioorthogonal fluorescent labeling, Institute of Organic Chemistry, Hungarian Academy of Sciences, Hungary
  • Tatsuo Shibata, Protein synthesis kinetic modeling in code expansion, RIKEN, Japan
  • Jean Lehmann, ribozyme and genetic code evolution, University of Paris-Sud, France
  • David Bensimon, Single molecule imaging, ENS, France
  • Sophie Vriz, Homeoproteins and Cell Plasticity, Collège de France
  • Antoine Triller, Cell biology of synapse, IBENS, France 

Staff scientists

Graduate students

Master students

Other (volunteers, sabbatical...)

Publications

All the team's publications