Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells

Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted

D-Peptide and D-Protein technology: Recent advances, challenges, and opportunities

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in D-enantiomers facilitating mirror-image replication and transcription of L-DNA. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery on non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities

Preparing recombinant “Split AEP” for protein labeling

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity. Here, we report a novel preparation protocol, in which the cap and catalytically active core domains are produced as separate entities. The active enzyme can be obtained in two chromatographic steps, immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC), with no acid activation required, thereby shortening the purification procedure from at least 2 days to less than 6 h. In addition to the original C247A mutation which enhanced reaction with various amino nucleophiles, an extra D29E mutation was introduced to prevent self-cleavage, which led to noticeable improvements in homogeneity and activity of the enzyme. Indeed, the resulting “split AEP” (i.e., core domain of OaAEP1-D29E/C247A) exhibited improved catalytic efficiency constant (kcat/KM) that was found to be ∼3-fold higher than that of the original acid-activated counterpart (OaAEP1-C247A). Furthermore, we described a protein labeling protocol that couples the enzymatic reaction with an irreversible chemical transformation, thereby enabling high conversion of labeled protein with a lowered amount of reagent. Precisely, an alternative Asn-Cys-Leu (NCL) recognition sequence was used for substrate recognition. As the byproduct contains an N-terminal cysteine, it can be transformed into an inert 1,2 aminothiol motif by reacting with formylphenyl boronic acid (FPBA). Finally, the opportunities and challenges associated with the use of asparaginyl ligase are discussed

Observations optiques d'etoiles jeunes enfouies dans leurs nuages moleculaires progeniteurs

SIGLEINIST T 75684 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc