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

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.

An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates.

The XpressCF+ cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+ reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant expression system, engineered to have an oxidizing cytoplasm.

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones.

Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins.

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies.

Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars.

Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars.

Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.

[Image: see text] Regions of the tyrosyl tRNA thought to interact with elongation factor Tu were randomized, and the resulting tRNA libraries were subjected to evolution. The tRNAs identified resulted in significantly improved unnatural amino acid-containing protein yields. In some cases, the degree of improvement varied in an amino acid-dependent manner.

Evolution of proteins with genetically encoded "chemical warheads".

We recently developed a phage-based system for the evolution of proteins in bacteria with expanded amino acid genetic codes. Here we demonstrate that the unnatural amino acid p-boronophenylalanine (BF) confers a selective advantage in the evolution of glycan-binding proteins. We show that an unbiased library of naive antibodies with NNK-randomized V(H) CDR3 loops converges upon mutants containing BF when placed under selection for binding to a model acyclic amino sugar.

A facile system for encoding unnatural amino acids in mammalian cells.

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E.

Efforts toward the direct experimental characterization of enzyme microenvironments: tyrosine100 in dihydrofolate reductase.

State secrets: Site-specific deuteration and FTIR studies reveal that Tyr100 in dihydrofolate reductase plays an important role in catalysis, with a strong electrostatic coupling occurring between Tyr100 and the charge that develops in the hydride-transfer transition state (see picture, NADP(+) purple, Tyr100 green). However, relaying correlated motions that facilitate catalysis from distal sites of the protein to the hydride donor may also be involved.

The incorporation of a photoisomerizable amino acid into proteins in E. coli.

An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in E. coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter.