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AssigneeMERZ PHARMA GmbH & CO. KGaA
InventorsJurgen Frevert, Michael Schmidt, Fred Hofmann, Gerhard Groer
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This invention relates to a novel method for manufacturing and obtaining recombinant proteins, such as clostridial neurotoxins, harbouring an N-terminal lysine from precursor proteins. The method comprises the step of expressing a nucleic acid sequence encoding a precursor protein comprising an N-terminal motif, which can be recognised by an endoprotease specific for a lysine in P′1 position, and the step of cleaving the precursor protein with the endoprotease. The invention further relates to novel precursor proteins used in such methods, nucleic acid sequences encoding such precursor proteins and novel recombinant proteins, such as clostridial neurotoxins, harbouring an N-terminal lysine.
1-28. (canceled)
A method for the generation of a recombinant protein with an N-terminal lysine comprising a step of contacting a precursor protein comprising an terminal motif motif X-Lys-linker with an endoprotease which specifically cleaves between X and Lys of the motif, wherein X is an endoprotease recognition sequence, and wherein the linker comprises at least three amino acid residues comprising (i) at least a second Lys residue and/or a Thr residue, and (ii) at least two consecutive Gly residues.
A precursor protein comprising an N-terminal motif X-Lys-linker, wherein X is an endoprotease recognition sequence, and wherein the linker comprises at least three amino acid residues comprising (i) at least a second Lys residue and/or a Thr residue, and (ii) at least two consecutive Gly residues.
A recombinant protein, wherein the N-terminus of the recombinant protein consists of the sequence Lys-linker, wherein the linker comprises at least three amino acid residues comprising (i) at least a second Lys residue and/or a Thr residue, and (ii) at least two consecutive Gly residues, and wherein the recombinant protein comprises at least 50 amino acid residues, at least 100 amino acid residues, or at least 200 amino acid residues.
Dependencies are inferred from the claim text.