Enhanced yield of recombinant proteins with site-specifically incorporated unnatural amino acids using a cell-free expression system.

Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems.

Tetracycline repressor-based mammalian two-hybrid systems.

The study of protein-protein interactions is critical for the understanding and regulation of biological systems. To that end, yeast two-hybrid systems have been used to study protein-protein interactions in vivo, but they frequently suffer from a high incidence of false positives when applied to mammalian systems. A novel mammalian two-hybrid system has recently been developed which exhibits lower background and higher sensitivity than earlier mammalian two-hybrid systems.

Femtosecond laser-assisted optoporation for drug and gene delivery into single mammalian cells.

In this work, focused near-infrared (NIR) femtosecond laser pulses were used to transiently perforate the cellular membrane of targeted human embryonic kidney (HEK) cells and the uptake of extrinsic molecules into the targeted cells was observed. Various cellular responses to the laser treatments were closely analyzed to optimize several experimental parameters such as laser power, exposure time and location of laser irradiation using a membrane impermeable fluorescent dye. The optimized parameters were used to investigate the entry of a plasmid DNA encoding green fluorescent protein (GFP) into the target cells.

Transforming a pair of orthogonal tRNA-aminoacyl-tRNA synthetase from Archaea to function in mammalian cells.

A previously engineered Methanocaldococcus jannaschii tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells.

A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker.

The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation, and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bioanalytical reagents. One approach to this problem is to incorporate a redox-active amino acid, 3,4-dihydroxy-l-phenylalanine (l-DOPA), that selectively forms a covalent linkage to the target protein.

Site-specific protein cross-linking with genetically incorporated 3,4-dihydroxy-L-phenylalanine.

Come together right now with L-DOPA: Chemical cross-linking is widely used to study protein-protein interactions. However, many cross-linking agents suffer from low reactivity or selectivity. An efficient and selective reaction of site-specific protein cross-linking was achieved using genetically incorporated 3,4-dihydroxy-L-phenylalanine.