Transforming a Pair of Orthogonal tRNA-aminoacyl-tRNA Synthetase from Archaea to Function in Mammalian Cells

A previously engineered Methanocaldococcus jannaschii –tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting -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

ENDOR Spectroscopy and DFT Calculations: Evidence for the Hydrogen-Bond Network Within α2 in the PCET of E. coli Ribonucleotide Reductase

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y[subscript 122]•) cofactor required to generate a thiyl radical (C[subscript 439]•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y[subscript 122] ⇆ W[subscript 48][?] ⇆ Y[subscript 356] in β2, across the subunit interface to Y[subscript 731] ⇆ Y[subscript 730] ⇆ C[subscript 439] in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y[subscript 730] in α2, and this mutant was incubated with β2, cytidine 5′-diphosphate, and adenosine 5′-triphosphate to generate a NH2Y730• in D2O. [[superscript 2]H]-Electron–nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C[subscript 439] and Y[subscript 731]. A third proton was assigned to a water molecule in close proximity (2.2 Å O–H···O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH[subscript 2]Y[subscript 730]• are consistent with the combined effect of the hydrogen bonds to Cys[subscript 439] and Tyr[subscript 731], both nearly perpendicular to the ring plane of NH[subscript 2]Y[subscript 730]. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available.National Institutes of Health (U.S.) (NIH GM29595

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

Genetic Engineering of Enzymes and Electrode Modifications for Biosensing Applications

Biosensing efficiency, selectivity and sensitivity rely first and foremost on a successful interfacing between enzymes and sensing interfaces. [...