Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

Provided herein are novel materials and methods for site-specific incorporation of phosphotyrosines into proteins. The novel methods of the invention encompass the use of a novel aminoacyl tRNA synthetase capable of charging compatible tRNAs with a phosphotyrosine precursor. The phosphotyrosine precursor is then incorporated, site-specifically, into a protein at sites where phosphotyrosine residues are desired. The phosphotyrosine precursors are subsequently treated to convert them into phosphotyrosine residues, yielding proteins with phosphotyrosines at selected sites. The scope of the invention encompasses novel aminoacyl tRNA synthetases, novel phosphotyrosine precursors, and methods of using these materials to create site-specific phosphorylated tyrosine residues in a protein.

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.

Provided herein are, inter alia, compounds of Formula (I): biomolecules (e.g., proteins, lipids, RNA, glycans) comprising the compounds; bioconjugates comprising the compounds; processes for preparing the compounds, biomolecules, and bioconjugates; and their uses.

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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.

The invention relates to a method of preparing engineered target polypeptides (TP) comprising in its amino acid sequence one or more, identical or different, non-canonical amino acid (ncAA) residues, by expressing said TP in an insect cell line (ICL) and by expressing novel orthogonal bacterial aminoacyl tRNA synthetase/tRNA pairs in said ICL; a baculoviral shuttle vector (bacmid) suitable or introducing the genetic information of said orthogonal tRNA synthetase/tRNA into said ILC; particular expression cassettes for expressing said particular tRNAs in said ILC; TPs obtained by said method; as we as a kit for preparing said TPs.

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.

Provided herein are compositions and methods for generating polypeptides using non-natural amino acids (nnAAs) and genetic machinery, wherein the modified polypeptides, such as therapeutic polypeptides, bind to albumin, such as serum albumin. Methods of substituting a non-natural amino acid in a first polypeptide to obtain a modified polypeptide, the nnAA in some instances comprising an albumin targeting group, are disclosed, as are methods for making populations of such modified polypeptides. A therapeutic polypeptide, interleukin-1 receptor antagonist (IL-1RA) is exemplified using the disclosed methods.