Cleavage of fusion proteins using Granzyme B protease
AssigneeHoffmann-La Roche Inc.
InventorsRikke Hoegh Lorentsen, Charlotte Harkjaer Fynbo
Life of this patent
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Abstract
A method for the preparation of a polypeptide of interest in authentic form by enzymatic cleavage of fusion proteins using Granzyme B protease (EC 3.4.21.79). There is also provided fusion proteins comprising a polypeptide of interest and a fusion partner, wherein the junction region between the polypeptide of interest and the fusion partner comprises a Granzyme B protease cleavage site adjacent to the polypeptide of interest, and a human Granzyme B protease variant wherein the Cystein residue no. 228 (chymotrypsinogen numbering) is mutated to Phenylalanine.
Claims24
A method for the preparation of a polypeptide of interest in authentic form, said method comprising the steps of:
- (i) providing a fusion protein comprising, from its N-terminal to its C-terminal, (a) a fusion partner, (b) a Granzyme B protease recognition site comprising a Granzyme B protease cleavage site that is cleavable by human Granzyme B protease, and wherein the recognition site comprises an amino acid sequence of the general formula
- P4 P3 P2 P1 ↓ (SEQ ID NO:
- 59)
- wherein
- P4 is amino acid I or V,
- P3 is amino acid E, Q or M,
- P2 is X, where X denotes any amino acid,
- P1 is amino acid D, and
- ↓ is said Granzyme B protease cleavage site, and
- (c) the polypeptide of interest, wherein said cleavage site is adjacent to the polypeptide of interest, and
- (ii) cleaving the fusion protein with Granzyme B protease at said cleavage site to yield said polypeptide of interest in authentic form.
A method of cleaving a fusion protein comprising contacting said fusion protein with the human Granzyme B protease variant wherein the Cystein residue no. 228 (chymotrypsinogen numbering) is mutated to Phenylalanine.
A method for the preparation of a polypeptide of interest in authentic form, said method comprising the steps of:
- (i) providing a fusion protein comprising, from its N-terminal to its C-terminal, (a) a fusion partner, (b) a Granzyme B protease recognition site comprising a Granzyme B protease cleavage site that is cleavable by human Granzyme B, wherein the recognition site comprises an amino acid sequence selected from the group consisting of ICPD↓ (SEQ ID NO:
- 61), IEAD↓(SEQ ID NO:
- 62), IEPD↓(SEQ ID NO:
- 63), IETD↓(-SEQ ID NO:
- 64), IQAD↓(SEQ ID NO:
- 65), ISAD↓(SEQ ID NO:
- 66), ISSD↓(SEQ ID NO:
- 67), ITPD↓(SEQ ID NO:
- 68), VAPD↓(SEQ ID NO:
- 69), VATD↓(SEQ ID NO:
- 70), VCTD↓(SEQ ID NO:
- 71), VDPD↓(SEQ ID NO:
- 72), VDSD↓(SEQ ID NO:
- 73), VEKD↓(SEQ ID NO:
- 74), VEQD↓(SEQ ID NO:
- 75), VGPD↓(SEQ ID NO:
- 76), VEID↓(-SEQ ID NO:
- 77), VRPD↓(SEQ ID NO:
- 78), VTPD↓(SEQ ID NO:
- 79), LEED↓(SEQ ID NO:
- 80), LEID↓(SEQ ID NO:
- 81), LGND↓(SEQ ID NO:
- 82), LGPD↓(SEQ ID NO:
- 83), and AQPD↓(SEQ ID NO:
- 84), and wherein ↓ is said Granzyme B protease cleavage site, and the polypeptide of interest, wherein said cleavage site is adjacent to the polypeptide of interest, and
- (ii) cleaving the fusion protein with Granzyme B protease at said cleavage site to yield said polypeptide of interest in authentic form.
Dependencies are inferred from the claim text.