Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Effects of transgenic expression of Brevibacterium linens methionine gamma lyase (MGL) on accumulation of Tylenchulus semipenetrans and key aminoacid contents in Carrizo citrange.

KEY MESSAGE: Carrizo transgenic plants overexpressing methionine-gamma-lyase produced dimethyl sulfide. The transgenic plants displayed more resistance to nematode attacks (Tylenculus semipenetrans) and may represent an innovative strategy for nematode control. Tylenchulus semipenetrans is a nematode pest of many citrus varieties that causes extensive damage to commercial crops worldwide. Carrizo citrange vr. (Citrus sinensis L. Usb × Poncirus trifoliate L. Raf) plants overexpressing Brevibacterium linens methionine-gamma-lyase (BlMGL) produced the sulfur volatile compound dimethyl sulfide (DMS). The aim of this work was to determine if transgenic citrus plants expressing BlMGL showed increased tolerance to T. semipenetrans infestation and to determine the effect on the content of key amino acids. While transgenic lines emitted dimethyl sulfide from leaves and roots, no sulfur-containing volatiles were detectable in wild-type Carrizo in the same tissues. Significant changes detected some key amino acids from leaves of transgenic plants such as aspartate, lysine, glycine, leucine and threonine with no changes in the amounts of methionine and α-ketobutyrate. In roots only glycine showed significant changes across all transgenic lines in comparison to wild-type plants. Transgenic plants expressing BlMGL and emitting DMS had less T. semipenetrans aggregation and more biomass than infected WT control plants, indicating that they may represent an innovative management alternative to pesticide/nematicide-based remedies.

A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus.

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/ E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.