Compositions are described for direct protein delivery into multiple cell types in the mammalian inner ear. The compositions are used to deliver protein(s) (such as gene editing factors) editing of genetic mutations associated with deafness or associated disorders thereof. The delivery of genome editing proteins for gene editing and correction of genetic mutations protect or restore hearing from genetic deafness. Methods of treatment include the intracellular delivery of these molecules to a specific therapeutic target.

The present invention provides a scFv comprising a heavy chain variable region (VH) and a light chain variable region linked by a first peptide linker, wherein an N-terminus and a C-terminus thereof are linked by a second peptide linker.

The present invention relates to a method for producing a conjugate of two substrates, the method comprising the steps of (a) providing two substrates, each substrate being independently selected from the one or more of the groups consisting of small molecules, and proteins, and (b) enzymatically conjugating the two substrates using a sortase F enzyme, or a catalytic domain thereof. One substrate comprises a sortase F recognition motif, while the other substrate comprises at least one motif selected from a Gly.sub.n motif, an Ala.sub.n motif, or a motif consisting of a mixture of Ala and Gly residues the motif totaling n residues, where n is an integer from ≥1 to ≤21. The reaction takes place in an aqueous reaction medium comprising a salt concentration of from about ≥0.01 to ≤3 M, thereby producing a conjugated product of the two substrates.

The present invention is directed generally to cell-targeted cytotoxic constructs comprising a targeting polypeptide, a linking polypeptide and a cytotoxic polypeptide. Preferably, (a) the targeting polypeptide is a R-spondin1 (RSPO1), R-spondin2 (RSPO2) or yoked chorionic gonadotropin (YCG), the linking polypeptide comprises LPXT (SEQ ID NO: 56) or NPXT (SEQ ID NO: 60) as well as others, where X is any amino acid, the linking polypeptide being positioned between the targeting ligand and (c) the cytotoxic moiety is an auristatin or a truncated serine protease, the serine protease having an IIGG (SEQ ID NO: 91), IVGG (SEQ ID NO: 92) or ILGG (SEQ ID NO: 93) at its N-terminus. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer).

The invention relates generally to methods for preparing recombinant nucleosomes. In particular, the invention relates to methods for ligating a histone peptide onto a fully assembled recombinant nucleosome. The invention further relates to modified core histone proteins, histone peptides to be ligated to the modified core histone proteins, and fully assembled recombinant nucleosomes and libraries of recombinant nucleosomes prepared by the methods of the invention.

The invention relates to surface modified extracellular vesicles, wherein the extracellular vesicles comprise an exogenous polypeptide tag that is covalently linked to a membrane protein of the extracellular vesicles. In a particular embodiment, the tag is covalently linked to the membrane protein of microvesicles by sortase-mediated ligation. Methods of preparing said extracellular vesicles and methods of using said extracellular vesicles loaded with therapeutic molecules for treating a disease are also disclosed herein.

There is provided a method for producing a peptide-nucleic acid complex containing a peptide and a nucleic acid encoding the peptide. The method for producing a peptide-nucleic acid complex includes a step of preparing a nucleic acid to which a transpeptidase N-terminal substrate motif has been added, the nucleic acid containing a first coding sequence encoding a peptide, a second coding sequence encoding a transpeptidase, and a third coding sequence encoding a transpeptidase recognition motif; a step of synthesizing a chimeric protein containing a domain of the peptide, a domain of the transpeptidase, and the transpeptidase recognition motif, from the nucleic acid to which the transpeptidase N-terminal substrate motif has been added, using a cell-free protein synthesis system; and a step of forming the peptide-nucleic acid complex by means of the transpeptidase domain.

Herein is reported a method for the enzymatic production of a polypeptide comprising the step of incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue), ii) a second polypeptide that has i) a glycinyl, an alaninyl, or a cysteinyl compound at its N-terminus, or ii) an oligoglycine, or oligoalanine, or a cysteine amino acid residue followed by one to three glycine or alanine amino acid residues at its N-terminus, or iii) a lysine amino acid residue within its 5 N-terminal amino acid residues, and iii) a third polypeptide with sortase A activity, in a deep eutectic solvent and thereby producing a polypeptide.

The present invention includes compositions and methods comprising sortase immune receptors and sortase chimeric antigen receptors (CARs).

Herein is reported a polypeptide comprising the amino acid sequence of SEQ ID NO: 38 as sole Listeria monocytogenes derived polypeptide and its use in conjugating polypeptides.