The object of the present invention is a method for the production of block polymers comprising a firstand a second block comprising the method steps: A) providing a first block having a nucleophilic peptide sequence for afirst transpeptidase enzyme, B) providing a second block having a peptide recognition sequence for the first transpeptidase enzyme, C) linking the first block to the second block by means of the first transpeptidase enzyme, wherein the first and second blocks are independently selected from nanoparticles, non-peptide polymers, and recombinant proteins. Such a production method makes it possible to build block polymers from identical or different blocks in a particularly simple and controlled manner.

The present invention relates to compositions and methods associated with antibody-bacterial effector translocase protein conjugates. Some aspects of the present invention relate to preventing or treating diseases using the antibody-protective antigen conjugates. Further aspects of the present invention relate to methods of chemically conjugating and synthesizing the antibody-bacterial effector translocase protein conjugates.

Herein is reported a method for producing an enzymatic conjugation product of three polypeptides comprising the simultaneous incubation of i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 20, wherein X can be any amino acid residue), a second polypeptide comprising the amino acid sequence LPXTA (SEQ ID NO: 31, wherein X can be any amino acid residue), a third polypeptide that has two N-termini whereby the polypeptide has an oligo-glycine G.sub.m (m=2 (SEQ ID NO: 22), or 3 (SEQ ID NO: 23), or 4 (SEQ ID NO: 24), or 5 (SEQ ID NO: 25)) amino acid sequence at its first N-terminus and an oligo-alanine A.sub.m (m=2 (SEQ ID NO: 26), or 3 (SEQ ID NO: 27), or 4 (SEQ ID NO: 28), or 5 (SEQ ID NO: 29)) amino acid sequence at its second N-terminus, a fourth polypeptide with sortase activity whereby the polypeptide is derived from Staphylococcus aureus sortase A, and a fifth polypeptide with sortase activity whereby the polypeptide is derived from Streptococcus pyogenes sortase A and the recovering of the conjugate from the reaction mixture.

Herein is reported a method for producing an enzymatic conjugation product of two polypeptides comprising incubating of a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 20, wherein X can be any amino acid residue), a second polypeptide has an oligo-alanine A.sub.m (m=2 (SEQ ID NO: 26), or 3 (SEQ ID NO: 27), or 4 (SEQ ID NO: 28), or 5 (SEQ ID NO: 29)) amino acid sequence at its N-terminus, a third polypeptide with sortase activity which is derived from Staphylococcus aureus Sortase A, and recovering the conjugate from the reaction mixture and thereby producing the enzymatic conjugation product of two polypeptides.

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.

Provided are improved methods for identifying the substrate recognition specificity or activity of a protease, convertase (sortase), or kinase. In some embodiments, methods are provided for identifying the endogenous protease or convertase cleaving patterns (e.g., “cleaveOme”) inside the secretory pathway of a living cell. Select embodiments involve aspects of yeast endoplasmic reticulum sequestration screening and next generation sequencing. Methods of producing polypeptides in Kex2 knockout yeast are also provided.

This application provides mutant sortase molecules and methods of making and using them. In a first aspect, disclosed herein, are sortase molecules having one or a combination of mutations. In an embodiment, a sortase molecule is optimized for a parameter of enzyme performance, e.g., Ca++ dependency (or independency) or reaction rate.

A deep eutectic solvent consisting of (2-hydroxyethyl) trimethyl ammonium chloride and dithiothreitol in a molar ratio of from 1:2 to 1:3 and from 0% to 10% co-solvent, and methods of enzymatic production of polypeptides using the deep eutectic solvent.

Cell-targeted cytotoxic agents, including sortase serine protease constructs, are provided. Such compounds can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer). In some aspects, recombinant sortase serine proteases, such as Granzyme B polypeptides, are provided that exhibit improved stability and cell toxicity.

Cell-targeted cytotoxic agents, including sortase serine protease constructs, are provided. Such compounds can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer). In some aspects, recombinant sortase serine proteases, such as Granzyme B polypeptides, are provided that exhibit improved stability and cell toxicity.