US2014221608A1PendingUtilityA1
Acid-cleavable linkers exhibiting altered rates of acid hydrolysis
Est. expiryNov 15, 2030(~4.3 yrs left)· nominal 20-yr term from priority
C07K 5/1021C07K 14/435C07K 2319/50C07K 2319/00C07K 7/06C07K 1/12C12P 21/06C07K 2319/01C12N 15/62
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Claims
Abstract
An acid-cleavable peptide linker comprising aspartic acid and proline residues is disclosed. The acid-cleavable peptide linker provides an altered sensitivity to acid-hydrolytic release of peptides of interest from fusion peptides of the formula PEP1-L-PEP2. The inventive linker, L, is described in various embodiments, each of which provides substantially more rapid acid-release of peptides of interest than does a single aspartic acid-proline pair. In an additional aspect, a method of increasing the stability of an acid cleavable linkage to acid hydrolysis is also provided.
Claims
exact text as granted — not AI-modified1 . A method of preparing at least one peptide of interest (“POI”) from a fusion peptide comprising at least one POI, comprising:
a) providing a recombinant cell synthesizing the fusion peptide of claim 21
b) contacting the fusion peptide with a solution of sufficiently acidic pH so that linker L is cleaved, and
c) isolating the at least one POI.
2 . (canceled)
3 . The method of claim 1 wherein the recombinant cell is a recombinant microbial cell.
4 . The method of claim 3 wherein the recombinant microbial cell is a recombinant yeast cell.
5 . The method of claim 3 wherein the recombinant microbial cell is a recombinant bacterial cell.
6 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 1 to about pH 4.
7 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 2 to about pH 4.
8 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 3 to about pH 4.
9 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH of about 4.
10 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 40° C. to about 90° C.
11 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 50° C. to about 80° C.
12 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 60° C. to about 70° C.
13 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 60° C.
14 . The method of claim 1 wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH of about pH 2 to about pH 4 using a temperature of about 50° C. to about 80° C.
15 . The method of claim 1 , wherein PEP1 and PEP2 are both POIs.
16 . The method of claim 15 , wherein the fusion peptide is soluble in the recombinant cell.
17 . The method of claim 15 , wherein the fusion peptide is insoluble in the recombinant cell.
18 . The method of claim 17 , wherein cleaving the fusion peptide under acidic conditions renders the at least one POI soluble.
19 . The method of claim 1 , wherein either PEP1 or PEP2 of the fusion peptide comprises an inclusion body tag, thereby comprising a non-POI portion of the fusion peptide.
20 . The method of claim 19 , wherein the non-POI portion remains insoluble after cleaving the fusion peptide.
21 . A fusion peptide comprising two peptides separated by an acid-cleavable linker according to the following general formula:
PEP1-L-PEP2
wherein,
a) PEP1 and PEP2 are independently functional peptides wherein at least one is a peptide of interest (“POI”); and
b) L is an acid-cleavable linker comprising a peptide selected from the group consisting of:
(SEQ ID NO: 1)
A. DPDP,
(SEQ ID NO: 2)
B. DPDPDP,
and
(SEQ ID NO: 3)
C. DPDPDPDP,
wherein D is aspartic acid and P is proline.
22 . (canceled)
23 . The fusion peptide of claim 21 wherein PEP1 and PEP2 are nonidentical.
24 . The fusion peptide of claim 23 , wherein the fusion peptide is soluble in a recombinant cell.
25 . The fusion peptide of claim 24 wherein the recombinant cell is a recombinant microbial cell.
26 . The fusion peptide of claim 25 wherein the recombinant microbial cell is a recombinant bacterial cell.
27 . The fusion peptide of claim 25 wherein the recombinant microbial cell is a recombinant yeast cell.
28 . The fusion peptide of claim 23 , wherein the fusion peptide is insoluble in a recombinant cell.
29 . The fusion peptide of claim 28 wherein the recombinant cell is a recombinant microbial cell.
30 . The fusion peptide of claim 29 wherein the recombinant microbial cell is a recombinant yeast cell.
31 . The fusion peptide of claim 30 wherein the recombinant microbial cell is a recombinant bacterial cell.
32 . The fusion peptide of claim 23 wherein either of PEP1 or PEP2 comprises an inclusion body tag (“IBT”).
33 . The fusion peptide of claim 32 wherein the fusion peptide is present in inclusion bodies.
34 . The fusion peptide of claim 23 wherein the acid-cleavable linker is cleaved by incubation at a pH in the range from about pH 1 to about pH 4.
35 . The fusion peptide of claim 23 wherein the acid-cleavable linker is cleaved by incubating at a temperature of about 40° C. to about 90° C.
36 . The fusion peptide of claim 35 wherein the acid-cleavable linker is cleaved by incubating at a temperature of about 50° C. to about 80° C.
37 . The fusion peptide of claim 36 wherein the acid-cleavable linker is cleaved by incubating at a temperature of about 60° C. to about 70° C.
38 . The fusion peptide of claim 23 wherein the acid-cleavable linker is cleaved by incubating at a pH of about pH 2 to about pH 4 and at a temperature of about 50° C. to about 80° C.
39 . A recombinant cell expressing a fusion protein having the structure
PEP1-L-PEP2 wherein, i) PEP1 and PEP2 are independently functional peptides, one of which is a POI; and ii) L is an acid-cleavable linker comprising a peptide selected from the group consisting of:
(SEQ ID NO: 1)
A. DPDP,
(SEQ ID NO: 2)
B. DPDPDP,
and
(SEQ ID NO: 3)
C. DPDPDPDP,
wherein D is aspartic acid and P is proline; and
wherein the expressed fusion peptide is present in the recombinant cell.
40 . (canceled)
41 . The recombinant cell of claim 39 wherein the recombinant cell is a recombinant microbial cell.
42 . The recombinant cell of claim 41 wherein the recombinant cell is a recombinant bacterial cell.
43 . The recombinant cell of claim 41 wherein the recombinant cell is a recombinant microbial cell is a recombinant yeast cell.
44 . An acid-cleavable peptide linker comprising a peptide selected from the group consisting of:
(SEQ ID NO: 1)
A. DPDP,
(SEQ ID NO: 2)
B. DPDPDP,
and
(SEQ ID NO: 3)
C. DPDPDPDP,
wherein D is aspartic acid and P is proline.
45 - 46 . (canceled)Join the waitlist — get patent alerts
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