| 摘要 |
This invention relates to a method of hydrolysis of the peptide bond between R1 and B in a specific designed amino acid sequence R1BXJZR3R2, where R1 represents a polypeptide of interest, R2 represents a sequence capable of specific binding to another component or molecule or another domain which needs to be cleaved, R3 represents an optional short peptide sequence, B represents a residue capable of accepting an acyl group, J represents a residue capable of metal ion binding, and X and Z represent amino acid residues, wherein the said method is based on a novel molecular mechanism of peptide bond hydrolysis, occurring in a specific complex of this metal ion with the BXJZ sequence. This method can be used to remove BXJZR3R2 domains in recombinant polypeptides, such as sequences capable of specific binding to another component or molecule to yield pure, unmodified R1 polypeptides of interest. The intermediate hydrolysis product can be reacted with other compounds to obtain derivatives of polypeptides of interest modified covalently at the C-terminus. |
| 主权项 |
1. A method of hydrolysis of a peptide bond between a polypeptide sequence R1 and an amino acid residue B wherein a recombinant protein represented by a formula R1BXJZR3R2 is designed and obtained by molecular biology methods which contains, starting from the N-terminus: an amino acid sequence of a target fusion protein comprising polypeptide sequence R1, a metal cation binding sequence BXJZ, and a functional domain R3R2, wherein:
R1 is a polypeptide sequence to be hydrolyzed; R2 is a polypeptide sequence to be removed; R3 represents a single peptide, a dipeptide, a polypeptide sequence or a covalent bond; B is an amino acid residue selected from the group consisting of Ser, Thr or a synthetic amino acid containing an alcohol moiety; X is an amino acid selected from the group consisting of Arg, Lys and His; J is an amino acid residue selected from the group consisting of Asp, Glu, Tyr, Met, Cys, and His, or J has a side chain bearing a group Yj, wherein Yj is a salt of an inorganic acid moiety or an organic acid moiety that forms an ionic bond with a metal ion; and Z is an amino acid residue, occurring naturally or synthetic, containing the —NH— group, which forms a peptide bond having formula —C(O)—NH— together with amino acid residue J;wherein the target protein R1 is released by contacting the recombinant protein with a metal ion Mn+ in a buffered solution for a time and under conditions effective to achieve total hydrolysis of the peptide bond between R1 and B, the metal ion M+n being Ni2+; wherein the method, the sequence BXJZ does not comprise SHHK or SAHK. |
