The present invention relates to methods and cross-linkers for the macrocyclization of proteins. The invention is useful for increasing the stability of a protein.

Herein is reported a method for the enzymatic production of a thioester comprising incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue), ii) a second polypeptide that has at its N-terminus a cysteine amino acid residue or is a cysteinyl compound, and iii) a third polypeptide with sortase A activity.

This invention relates to a method of producing a circularised form of a target protein from a fusion protein and to a fusion protein capable of producing a circularised form of a target protein. The fusion protein can comprise: the target protein; at least one circularisation site adjacent the target protein; and an enzyme domain capable of interacting with the at least one circularisation site and circularising the target protein. The target protein can be a membrane scaffold protein or a cyclotide such as SFTI, Vc1.1, Kalata B1 or MCOTI-II.

Recombinant microorganisms and methods of using same. The recombinant microorganisms include one or more modifications that reduce the expression and/or activity of a sortase, a sortase-dependent protein, a fibronectin-binding protein, an autolysin, a surface-layer protein, an aggregation-promoting factor, and/or a collagen-binding protein. The modifications can reduce the adhesion characteristics with respect to the non-modified microbes. The recombinant microorganisms can further include a recombinant gene configured to express a biologic. The recombinant microorganisms can be used as delivery vehicles to deliver the biologics to sites such as the gastrointestinal tract.

The present disclosure relates to the field of biotechnology. In particular, provided are a ligase fusion protein and an immobilized ligase comprising the same. Also provided is use of the ligase fusion protein or the immobilized ligase in the preparation of conjugates. Further provided is a process for the preparation of conjugates using a ligase or a ligase unit.

Herein is reported a method for producing an antibody Fc-region conjugate comprising as first component an antibody Fc-region and as second component at least one binding entity that specifically binds to a target using a transpeptidase for enzymatic conjugation of the antibody Fc-region to at least one binding entity.

Herein is reported a method for producing a bispecific antibody comprising the step of incubating (i) an antibody Fab fragment or a scFv antibody comprising within the 20 C-terminal amino acid residues the amino acid sequence LPX1TG (SEQ ID NO: 01), (ii) a one-armed antibody comprising a full length antibody heavy chain, a full length antibody light chain, and an Fc-heavy chain, whereby the full length antibody heavy chain and the full length antibody light chain are cognate antibody chains that thereof forms an antigen binding site, whereby the full length antibody heavy chain and the Fc-heavy chain are covalently linked to each other via one or more disulfide bonds forming an antibody hinge region, and whereby the Fc-heavy chain has an oligoglycine amino acid sequence at its N-terminus, and (iii) a Sortase A enzyme.

A conjugation device includes at least one flow reactor having an inlet and an outlet, the flow reactor(s) being completely filled with a support such as a matrix including 1) chromatography beads, fibers or membranes, and 2) a biologic catalyzer, namely the enzyme ligase, which is immobilized onto this support; a fluid delivery unit in fluid communication with the inlet of the flow reactor(s) and configured to continuously provide the flow reactor(s) with at least one kind of reaction fluid such as antibody and linker-payload according to stages of the conjugation process, the at least one kind of process fluid including a first moiety and a second moiety of a conjugate to be produced; and a fluid collection unit in fluid communication with the outlet of the flow reactor(s) and configured to control collection of fluid flowing out of the outlet of the flow reactor(s) according to the stages of the conjugation process. In a period of enabling the at least one kind of reaction fluid to continuously flow through the flow reactor(s), a conjugation reaction is conducted between the first moiety and the second moiety under catalysis of the ligase to produce the conjugate.

The present disclosure relates to the field of biotechnology. In particular, provided are a ligase fusion protein and an immobilized ligase comprising the same. Also provided is use of the ligase fusion protein or the immobilized ligase in the preparation of conjugates. Further provided is a process for the preparation of conjugates using a ligase or a ligase unit.

The present disclosure relates to the field of biotechnology. In particular, provided are a ligase fusion protein and an immobilized ligase comprising the same. Also provided is use of the ligase fusion protein or the immobilized ligase in the preparation of conjugates. Further provided is a process for the preparation of conjugates using a ligase or a ligase unit.