ABSTRACT
The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNAPyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/genetics , Flow Cytometry , High-Throughput Screening Assays , Lysine/analogs & derivatives , Protein Engineering , Amino Acyl-tRNA Synthetases/metabolism , Flow Cytometry/methods , Gene Library , Lysine/chemistry , Lysine/metabolism , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The biotin-streptavidin interaction is among the strongest known in nature. Herein, the site-directed incorporation of biotin and 2-iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. By using the high-affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well-defined protein-drug conjugates.
Subject(s)
Biotin/analogs & derivatives , Lysine/analogs & derivatives , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Biotin/genetics , Biotin/metabolism , Biotinylation , Escherichia coli/metabolism , HEK293 Cells , Humans , Lysine/genetics , Lysine/metabolism , Methanosarcina barkeri/enzymology , Mutation , Protein Binding , Protein Processing, Post-Translational , Rec A Recombinases/chemistry , Rec A Recombinases/metabolism , Streptavidin/metabolismABSTRACT
Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl-methyl-ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo- and thermoadaptation.
Subject(s)
Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/isolation & purification , Salts/metabolism , Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Indian Ocean , Salts/chemistry , TemperatureABSTRACT
Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand. Light scattering is an established process to measure virus particles' size, though due to cell impurities, adequate quantification of adenovirus particles in cell lysates by light scattering has been impossible until today. This report describes a new method using light scattering to measure virus concentration in nonpurified cell lysates. Here we report application of light scattering, a routine method to measure virus particle size, to virus quantification in enzymatically conditioned crude lysates. Samples are incubated with phospholipase A2 and benzonase and filtered through a 0.22 µm filter cartridge prior to quantification by light scattering. Our results show that this treatment provides a precise method for fast and easy determination of total adenovirus particle numbers in cell lysates and is useful to monitor virus recovery throughout all downstream processing.
