Expanding The Genetic Code with Halogen Bonding and Enhancing the Membrane Transport of Proteins in Mammalian Cells

                                                     Department of Biological Sciences & Engineering


Venue: Room 303, Samgata, Nila campus

TIme: 3:30 PM


Expanding The Genetic Code with Halogen Bonding and Enhancing the Membrane Transport of Proteins in Mammalian Cells


Dr Surendar Reddy Jakka

Dept. of Inorganic & Physical Chemistry, Indian Institute of Science, Bengaluru


Direct delivery of proteins into mammalian cells is a challenging problem in biological and
biomedical applications. The most common strategies for the delivery of proteins into the cells include the
use of cell-penetrating peptides or supercharged proteins. In this talk, I will be presenting that a single
atom change, hydrogen to halogen, at one of the tyrosine residues can increase the cellular entry of ~28.0
kDa green fluorescent protein (GFP) in mammalian cells. The substitution of iodine/halogen was
achieved by using the genetic code expansion (GCE) methodology, in which an evolved orthogonal
Methanococcus jannaschii (Mj) tyrosyl-tRNA synthetase/tRNA pair for 3-iodo L-tyrosine as substrate
and amber codon as unique codon to decode during co-translational insertion. Upon co-incubation of the
halogenated GFPs with mammalian cells, the halogen dependent uptake was observed, and highest uptake
was observed for the iodinated GFP. The mechanistic investigations revealed the halogenated protein uptake is facilitated by a receptor-mediated endocytosis and the cargo can be released effectively into cytosol by co-treatment with the endosomolytic peptide ppTG21.


A schematic representation of caveolae-mediated cellular uptake and proposed endosomal
escape route for iodinated/halogenated GFP

Next, in order to incorporate 3,5-dihalo-L-tyrosines, a rational design of Mj tyrosyl-tRNA/tRNA
pair was performed based on the halogen bonding as the guiding principle and successfully identified
mutants that can charge the tRNA with 3,5-diiodo-L-tyrosine. For the first time, a mutant tRNA
synthetase with strong preference to charge the tRNA with 3,5-diiodo-L-tyrosine as compared to that of
3,5-dichloro-, and 3,5-dibromo-L-tyrosine has been developed. Finally, I will be talking briefly about in
vivo continues evolution strategies in terms of evolving orthogonal tRNA synthetase/tRNA pairs for
multisite incorporation of unnatural amino acids at defined positions in protein of interest and future

1. Wang, L., Brock, A., Herberich, B., Schultz, P. G. Expanding the genetic code of Escherichia coli
Science, 2001, 292, 498-500.
2. Sakamoto, K., et al. Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength
anomalous dispersion phasing in protein crystallography Structure. 2009, 17, 335-344.
3. Jakka, S. R., Govindaraj. V., Mugesh, G. A single atom change facilitates the membrane transport
of green fluorescent proteins in mammalian cells Angew. Chem. Int. Ed. 2019, 58, 7713-7717.
4. Govindaraj, V., Ungati, H., Jakka, S., R., Bose, S., Mugesh, G. Directing traffic: halogen bond-
mediated membrane transport Chem. Eur. J., 2019, 25, 11180-11192.

5. S. R. Jakka, Reddy, K. M, S. Jaiswal, G. Mugesh, Expanding the Genetic Code by Halogen Bonding.
(under review).