Provided herein are modified amino acids comprising a tetrazine groups according to Formula I: polypeptides, antibodies, payloads and conjugates comprising these modified amino acid residues derived from the modified amino acids, and methods of producing the polypeptides, antibodies, payloads and conjugates comprising the modified amino acid residues. The polypeptides, antibodies, payloads and conjugates are useful in methods of treatment and prevention, methods of detection and methods of diagnosis. ##STR00001##

A method for secretory production of a protein containing a noncanonical amino acid is provided. Secretory production of a noncanonical amino acid-containing protein is carried out by culturing a coryneform bacterium having a genetic construct for secretory expression of a protein containing a noncanonical amino acid, which is modified to express an orthogonal pair of tRNA corresponding to the noncanonical amino acid and aminoacyl tRNA synthetase.

The invention provides aminoacyl-tRNA synthetases, compositions thereof, and methods for use thereof.

The invention provides methods and compositions for a mutein aminoacyl-tRNA synthetase that preferentially charges a tRNA with a non-natural amino acid. Also provided are methods for incorporating the non-natural amino acids, pyridinyl-amino tetrazine amino acids, into a protein, and further conjugating a biologically active adduct to the pyridinyl-amino tetrazine.

The present invention relates to a method for producing eukaryotic cell lysates for cell-free protein synthesis comprising at least one exogenous enzyme, wherein the method comprises at least the following steps: a) providing eukaryotic cells transfected with at least one donor template coding for at least one exogenous enzyme which is selected from the group comprising orthogonal aminoacyl-tRNA synthetases and viral RNA polymerases: b) cultivating the transfected cells of step a) for a predetermined period of time and subsequently harvesting the cells; and c) disrupting the harvested cells and preparing a cell lysate for cell-free protein synthesis therefrom. More specifically, the invention relates to a method for producing eukaryotic cell lysates which are capable of cell-free synthesis of a target protein comprising a non-canonical amino acid, wherein the method comprises at least the following steps: a) providing eukaryotic cells transfected with at least one donor template coding for an orthogonal aminoacyl-tRNA synthetase. i.e. an aminoacyl-tRNA synthetase which is specific for a tRNA which is not recognized by endogenous aminoacyl-tRNA synthetase in said eukaryotic cells and is specific for a corresponding non-canonical amino acid: b) cultivating the transfected cells of step a) for a predetermined period of time and subsequently harvesting the cells; and c) disrupting the harvested cells and preparing a cell lysate therefrom. Further aspects of the present invention relate in particular to eukaryotic cell lysates obtainable by the above methods as well as to a method for performing cell-free synthesis of a target protein comprising a non-canonical amino acid.

This disclosure provides engineered tRNAs and the corresponding aminoacyl tRNA synthetase for efficient production of proteins containing non-standard amino acids. These engineered orthogonal tRNA (O-tRNA)/orthogonal aminoacyl tRNA synthetase (O-RS) pair, i.e., Orthogonal Translation Systems (OTSs) can be used to incorporate a non-standard amino acid in a specific position in a growing polypeptide in response to a selector codon that is recognized by the engineered tRNA.

The invention relates generally to engineered antibodies containing unnatural amino acids (UAAs) and methods of making and using such antibodies.

This invention relates to an unnatural amino acid-based ionic liquid, and preparation methods and applications thereof. It specifically provides a combination for preparing a protein comprising an unnatural amino acid, comprising: (1) one or more aminoacyl-tRNA synthetases capable of binding to a mutated tRNA; (2) one or more mutated tRNAs with an anti-codon loop mutated to complement a termination codon; (3) various unnatural amino acid-based ionic liquids. The combination can be used for recombinant expression of a target protein comprising an unnatural amino acid. The unnatural amino acid-based ionic liquids can improve the read-through efficiency of the genetic codon expansion system for a premature termination codon (PTC) and/or the incorporation efficiency of unnatural amino acids.

The invention provides methods and compositions for a mutein aminoacyl-tRNA synthetase that preferentially charges a tRNA with a non-natural amino acid. Also provided are methods for incorporating the non-natural amino acids, pyridinyl-amino tetrazine amino acids, into a protein, and further conjugating a biologically active adduct to the pyridinyl-amino tetrazine.

The present disclosure provides an engineered cell comprising a sulfotransferase. In some embodiments, the sulfotransferase is NnSULT1C1 sulfotransferase. The present disclosure also provides methods for the biosynthesis of a peptide containing at least one sulfotyrosine residue, as well as compositions comprising a peptide containing at least one sulfotyrosine residue. In some embodiments, the compositions and methods of the present disclosure provide therapeutic peptides for use in treating and/or preventing a disease or disorder, such as an HIV-1 infection.