Therapeutic Applications of an Expanded Genetic Code

Abstract

ω‐Transaminases are a valuable class of enzymes for the production of chiral amines with either (R)‐ or (S)‐configuration in high optical purity and 100% yield by the biocatalytic reductive amination of prochiral ketones. A versatile new assay was developed to quantify ω‐transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymes based on the conversion of 1‐(6‐methoxynaphth‐2‐yl)alkylamines. The associated release of the acetonaphthone product can be monitored by the development of its bright fluorescence at 450 nm with very high sensitivity and selectivity. The assay principle can be used to quantify ω‐transaminase catalysis over a very broad range of enzyme activity. Because of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases. For assay substrates that incorporate structural variations, an efficient modular synthetic route was developed. This includes racemate resolution by lipase‐catalyzed transacylation to furnish enantiomerically pure (R)‐ and (S)‐configured amines. The latter are instrumental for the rapid enantioselectivity typing of ω‐transaminases. This method was used to characterize two novel (S)‐selective taurine‐pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

The use of unnatural amino acids to study and engineer protein function

“…The expanded chemical repertoire provided by UAAs facilitates engineering therapeutically important proteins such as antibodies and hormones [42]. For example, bispecific antibodies have been produced that induce cytotoxicity by crosslinking HER2+ and CD3+ antigens, prevalent on cancer cells and cytotoxic T lymphocytes, respectively (Figure 3a) [43 ].…”

A platform for constructing, evaluating, and screening bioconjugates on the yeast surface

“…The biosynthetic production of proteins containing noncanonical amino acids (ncAAs) provides powerful approaches to solving many outstanding problems in biology and biomolecular engineering (Johnson et al, 2010;Sun et al, 2014) including the atomic-level dissection of protein biophysical properties (Kwon et al, 2010;Ye et al, 2010) and the construction of therapeutic leads (Frost et al, 2015;Horiya et al, 2014;Ng et al, 2015). Emerging efforts combining ncAAs with mRNA (Hayashi et al, 2012;Horiya et al, 2014), phage (Liu et al, 2008), or E. coli display show promise in the discovery of proteins with an expanded range of chemical functionality exhibiting improved biophysical and therapeutic characteristics.…”

A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation

“…1f,8 Development of engineered nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that selectively charge such ncAAs has enabled their facile site-specific incorporation into proteins expressed in living cells, which can be subsequently used to precisely attach a variety of entities, from biophysical probes to therapeutic agents. 1f,8,9 However, many of the available chemistries are limited by their slow kinetics, the need for toxic catalysts, or a lack of compatibility with other chemistries to allow the simultaneous attachment of multiple distinct entities at different sites. 10 Consequently, there is continued interest in additional genetically encoded chemical functionalities that can be rapidly and chemoselectively labeled using catalyst-free conjugation reactions, and that are also compatible with other available chemistries to allow concurrent labeling at multiple sites.…”

“…9,17 To evaluate if CRACR can be used to generate such antibody–conjugates in a site-specific manner, we expressed the previously described Fab fragment 18 of the anti-Her2 antibody Herceptin 19 in our engineered E. coli strain, site-specifically incorporating 5HTP at position 169. The wild-type Fab protein was also expressed as a control (lysine at position 169).…”