14 results about "Sortase" patented technology

Discussed herein are novel engineered polypeptides which are effective at cutting and tagging H3 histone tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of H3 histone modification crosstalk after treatment with different histone deacetylase inhibitors.

The present invention provides a method of transpeptidation, a transpeptidation product obtainable from the method, and a chimeric fusion polypeptide. An exemplary method of transpeptidation includes linking a donor substrate to a recipient protein or peptide using a transpeptidase, where the donor substrate is a protein, peptide, amino acid amide, or amino acid ester, and where the linking reaction produces a linking product and a by-product as a substrate for the transpeptidase; and cleaving one or more amino acids from the end of the by-product using an exopeptidase such that the truncated by-product is not a substrate for the transpeptidase, thereby moving the reaction equilibrium of the transpeptidation reaction to the ligation product.

A sortase variant with improved catalytic activity and stability, in particular said mutant further comprises the mutations M155V / L200F or M155V / K162P, according to SEQ ID NO: 1, with respect to the parent sortase (SEQ ID NO: 2). Also the use of said sortase mutant is involved.

Provided in the present invention are a fatty-acid-chain-modified GLP-1-FGF21 fusion protein, and a preparation method therefor and the use thereof. In the fusion protein, after a specific mutation site is introduced at the C-terminus of FGF21, the terminus of GLP-1-FGF21 is subjected to a fatty-acid-chain modification by using a Sortase-A enzyme-mediated molecular modification technique. The fatty-acid-chain modification can achieve: extended half-life of GLP-1-FGF21; slowed terminal degradation of FGF21 by fibroblast activation protease (FAP); and low-cost operation and rapid preparation of the GLP-1-FGF21 fusion protein in an aqueous solution at room temperature. The enzyme-mediated molecular modification technique is used on the GLP-1-FGF21 fusion protein of the present invention, thereby providing significant advantages.

The invention relates to the use of precursor peptides and B1 proteins from lasso peptide biosynthesis systems for affinity purification, proximity-based sortase-mediated protein purification and ligation, and detection of fusion proteins. For proximity-based sortase-mediated protein purification and ligation, the invention relates to techniques that link protein purification with conjugation to other agents, including therapeutic agents, imaging agents, or linkers.

The application provides a glutamyl transpeptidase mutant and application thereof. The glutamyl transpeptidase mutant comprises: (a) a protein which is mutated based on wild-type glutamyl transpeptidase shown in SEQ ID NO: 1, and the mutation comprises mutation at any one or more of the following positions: H87, Q89, D190, T193, E197, M323, T412, Q430, E548 or S552; or (b) a protein which has more than 70% homology with the amino acid sequence defined in (a) and has glutamyl transpeptidase activity. The application can solve the problem of low activity of glutamyl transpeptidase under acidic conditions in the prior art and is suitable for the technical field of microorganisms.

The invention discloses a Sortase A transpeptidase mutant and application thereof in the technical field of bioengineering, the Sortase A transpeptidase mutant is obtained through single mutation or double mutation on the basis of a protein sequence shown in SEQ ID NO.2, the protein sequence shown in SEQ ID NO.2 is a truncated mutant at the 60th-206th site made on wild type Sortase A transpeptidase shown in SEQ ID NO.1, and the Sortase A transpeptidase mutant is obtained through single mutation or double mutation on the basis of the protein sequence shown in SEQ ID NO.2. The Sortase A transpeptidase mutant provided by the invention has higher yield, activity and thermal stability, and has wider application conditions. Besides, a cysteine is introduced to the C terminal of the Sortase A transpeptidase mutant protein, and the Sortase A transpeptidase mutant is fixed on the magnetic beads by utilizing the principle of sulfydryl covalent coupling, so that the application value of the Sortase A transpeptidase mutant in protein labeling is realized.

Described herein is a protein display selection method which uncouples a protein of interest (POI) library from the display selection system. Display of the POI can be achieved by forming a covalent bond between the POI and the anchor protein post expression either by enzymatic protein ligation (e.g. SpyLigase, SnoopLigase, sortase, butelase, peptiligase etc.) or by spontaneous covalent bond formation (e.g. SpyTag / SpyCatcher, SnoopTag / SnoopCatcher, etc.). The POI library is fused to a tethering sequence, for example SpyTag, at the C-terminus of the POI which then forms a covalent bond to a capture sequence found on an anchor protein, for example, the SpyCatcher-fused anchor protein, e.g., a SpyCatcher-geneIII protein (SpyCatcher-pIII) fusion, for the most common form of phage display. Nucleic acid constructs, host cell systems and methods of producing the protein display systems are also provided.

In some embodiments, provided is an engineered Tn5 transposase complex, comprising: at least one Tn5 transposase, comprising a poly-glycine sequence; and at least one antibody that is covalently linked to the at least one Tn5 transposase by a sortase, wherein the at least one antibody that is specific to a target site of a chromatin sequence. Other example embodiments are described herein. In certain embodiments, provided engineered Tn5 transposase complexes, systems, methods and kits provide outstanding performance in identifying chromatin features especially co-occurrence of chromatin features in samples and enable identifying multiple chromatin features at single cell resolution.

A compound A-L-B, wherein A is a nanoparticle (NP)-forming unit, preferably a NP-forming polypeptide or a NP-forming protein, preferably a NP-forming polypeptide or a NP-forming protein having its C-terminus covalently bound to the N-terminus of L via a peptide bond; L is a polypeptide having the following amino acid sequence written in the single letter code z1–G X1 X2 G X3 G X4 X5 G X6 G X7 G–z2, wherein X1 = any amino acid except H, C, and W, X2 = any amino acid except H, C, and W, X3 = any amino acid except H, C, and W, X4 = any amino acid except H, C, and W, X5 = any amino acid except H, C, and W, X6 = any amino acid except H, C, and W, X7 = any amino acid except H, C, and W, z1 represents the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids, and z2 represents the C-terminus of the polypeptide, or a modification of the C- terminal carboxyl group of the polypeptide, which modification (i) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1 or -C(O)-NR2R3, wherein R1 is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C≡CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2 and R3 is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C≡CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or (ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or (iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond; wherein the side chain of each amino acid of the polypeptide L independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; and B is a compound having biological activity, preferably a protein, which preferably has its N-terminus covalently bound to the C-terminus of L via a peptide bond; wherein the N-terminus of L is covalently bound to A, preferably via a peptide bond, and wherein the C-terminus of L is covalently bound to B, preferably via a peptide bond.

The present invention relates to the provision of means and methods for the enzymatic ligation of peptides and / or proteins. In particular, herein provided are means and methods for the efficient coupling of peptides and / or proteins using transpeptidase enzymes belonging to the Connectase family, which are characterized by an N-terminal DUF2121 domain with an N-terminal serine or threonine residue. Also provided are, inter alia, compositions, polypeptides, and kits for carrying out the herein provided methods.

The present application relates to a sortase mutant having improved catalytic activity and thermal stability, and also relates to the use of the sortase mutant.

The present invention provides an efficient method for producing lipid molecules to which a ligand is bound. One aspect of the present invention provides molecules in which a ligand specific to a target cell and a lipid molecule are covalently bonded (hereinafter referred to as "targeted lipid molecules"), wherein a covalent bond (amide bond) is formed between a carboxy group of a threonine residue contained in a sortase recognition motif possessed by one of the ligand and the lipid molecule and a glycine residue or an amino group possessed by the other, and the glycine residue or the amino group is directly covalently bonded to the ligand or the lipid molecule. Another aspect of the present invention provides a production method for said targeted lipid molecules, the method comprising a step for reacting the ligand and the lipid in the presence of a sortase to covalently bond (amide bond) a carboxy group of a threonine residue contained in a sortase recognition sequence with the glycine residue or the amino group, wherein one of the ligand and the lipid molecule has the sortase recognition sequence and the other has the glycine residue or the amino group.

In some embodiments, an engineered Tn5 transposase complex is provided, the complex comprising: at least one Tn5 transposase comprising a polyglycine sequence; and at least one antibody covalently linked to the at least one Tn5 transposase via a sortase, wherein the at least one antibody is specific for a target site of a chromatin sequence. Other exemplary embodiments are described herein. In certain embodiments, the engineered Tn5 transposase complexes, systems, methods, and kits provided provide excellent performance in identifying chromatin features, particularly co-occurrence of chromatin features in a sample, and are capable of identifying multiple chromatin features at single cell resolution.