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. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.

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. It has successfully detected interactions with dissociation constants ranging from 0.99 nM to 55 µM. The system was built upon the tetracycline repressor-tetracycline operator interaction and is suitable for use in the study of most, if not all, mammalian protein-protein interactions.

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. Since laser beam with higher-than-threshold energy level will disintegrate cells, we used Matlab simulations to characterize the laser irradiance and free electron distribution caused by the femtosecond-optoporation process. The simulation results showed that the free electron distribution is much narrower than the laser irradiance, which implies that the transient perforation can even be smaller than the size of the laser focal volume. Femtosecond laser-assisted optoporation when combined with lab-on-a-chip devices can be useful in single cell-based high-throughput screening.

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. Thus we will be able to introduce a large array of possibilities for protein modifications 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. Such peptide-based probes can also be fused to tailored reporter proteins and easily expressed in bacterial cultures. As a demonstration, a candidate peptide, TOP1, that weakly binds to the target protein, the Src homology 3 (SH3) domain of human Abelson tyrosine kinase (Abl), was fused to green fluorescent protein (GFP) and l-DOPA was site-specifically incorporated into the peptide region (TOP1-DOPA-GFP). TOP1-DOPA-GFP produced from Escherichia coli was used in a Western blot-type experiment to show that the Abl SH3 domain can be detected in one step by observing the fluorescence. The molecular design presented in this work is significant in that the same approach could be used to transform many other protein-binding peptides with insufficient affinities into protein detection probes with a variety of fused reporter or therapeutic proteins.

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.