US2018087081A1PendingUtilityA1
Protein expression enhancing polypeptides
Est. expiryDec 5, 2032(~6.3 yrs left)· nominal 20-yr term from priority
A61P 43/00A61P 25/16A61P 25/28A61P 25/14A61P 25/00A61P 11/00C07K 16/244C07K 14/315C07K 2319/35C07K 16/241C07K 14/8125C12N 9/6437C07K 19/00C07K 2317/14C07K 14/31C07K 7/06C07K 14/5437C12P 21/02C07K 14/47C07K 2319/30C07K 14/755C07K 14/7155C12N 9/644C07K 2319/70C07K 2319/43A61K 48/00C07K 14/705C07K 14/01C07K 2319/705C07K 16/22C07K 2319/00C07K 7/08
37
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
Fusion proteins comprising a protein expression enhancing polypeptide linked to a target protein binding domain and nucleic acid molecules encoding such fusion proteins are described for use in enhancing expression and/or location of a targeted protein of interest, for restoring lost functions in cells, and for treating disease. Additional fusion proteins comprising a target protein of interest modified with a fusion partner comprising a protein expression enhancing polypeptide are also disclosed.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A fusion protein comprising:
(1) an isolated protein expression enhancing polypeptide (PEEP) domain linked to (2) a target protein of interest, wherein said isolated protein expression enhancing polypeptide domain (1) is selected from the group consisting of: (a) an isolated J domain of a J protein comprising a polypeptide domain of a J protein comprising four α helices, helices I, II, III, and IV, and having a histidine, proline, and aspartic acid sequence motif (HPD motif) between helix II and helix III, or an active fragment of said J domain; and (b) a J domain analog polypeptide comprises the amino acid sequence of formula I:
(I) X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO:47), wherein:
X1 is isoleucine (I), leucine (L), valine (V), alanine (A), or methionine (M);
X2 and X3 are each independently any amino acid with the proviso that one or both are K or R;
X4 is any amino acid or X4 may be absent when X1 through X3 are present and X5 through X9 are present;
X5 is tyrosine (Y), tryptophan (W), or phenylalanine (F);
X6 and X7 are each independently any amino acid with the proviso that one or both are lysine (K) or arginine (R); or either one of X6 and X7 may be absent when the other is K or R and when X1 through X5 are present and X8 and X9 are present; and
X8 and X9 are any amino acid with the proviso that one or both are leucine (L) or alanine (A); or one of X8 and X9 may be absent when the other is L or A and when X1 through X7 are present; and
wherein said fusion protein is expressed in a host cell at an enhanced level as compared to the level of expression of the unmodified target protein of interest.
2 . The fusion protein according to claim 1 , wherein the protein expression enhancing polypeptide domain (1) is an isolated J domain of a J protein having an amino acid sequence selected from any one of SEQ ID NOs:1-46.
3 . The fusion protein according to claim 1 , wherein the protein expression enhancing polypeptide domain (1) comprises an amino acid sequence selected from the group consisting of:
(SEQ ID NO: 48)
I-K-K-A-Y-R-K-L-A,
(SEQ ID NO: 49)
I-K-K-A-Y-K-L-A-L-Q,
(SEQ ID NO: 50)
I-K-K-A-Y-R-L-A-L-Q,
(SEQ ID NO: 51)
I-K-K-A-Y-R-K-A-L-Q,
(SEQ ID NO: 52)
I-K-K-A-Y-R-K-L-L-Q,
(SEQ ID NO: 83)
I-R-K-A-Y-R-K-L-S-L-T-L,
(SEQ ID NO: 84)
I-K-K-Q-Y-R-L-L-S-L-K-Y,
(SEQ ID NO: 85)
I-K-K-A-F-H-K-L-A-M-K-Y,
(SEQ ID NO: 86)
I-R-Q-A-F-K-K-L-A-L-K-L,
(SEQ ID NO: 87)
I-I-K-A-Y-R-K-L-A-L-Q-W,
(SEQ ID NO: 88)
I-A-R-A-Y-R-Q-L-A-R-R-Y,
(SEQ ID NO: 89)
I-K-R-A-Y-R-R-Q-A-L-R-Y,
(SEQ ID NO: 90)
I-K-K-S-Y-R-K-L-A-L-K-Y,
and
(SEQ ID NO: 91)
I-K-K-A-Y-K-R-L-A-M-K-Y.
4 . The fusion protein according to claim 1 , wherein the protein expression enhancing polypeptide domain (1) is an isolated J domain of an Erdj protein selected from the group consisting of: Erdj1, Erdj2, Erdj3, Erdj4, Erdj5, Erdj6, and Erdj7.
5 . The fusion protein according to claim 4 , wherein the protein expression enhancing polypeptide domain (1) is an isolated J domain of Erdj3 (SEQ ID NO:4).
6 . The fusion protein according to claim 1 , wherein the protein expression enhancing polypeptide domain (1) is a J domain analog polypeptide that comprises the amino acid sequence of formula I.
7 . The fusion protein according to claim 6 , wherein said J domain analog polypeptide comprises the amino acid sequence (SEQ ID NO:326) wherein:
X1 is I, L, V, A, or M; the dipeptide X2-X3 is selected from the group consisting of: KR, KK, RK, RR, AK, AR, KA, IK, NK, KQ, RQ, and RD; X4 is A, S, T, R, S, Q, E, F, C, or I; X5 is Y or F; the dipeptide X6-X7 is selected from the group consisting of: KR, KK, RK, RR, RQ, FR, RL, KL, HK, LK, QK, and KV; and the dipeptide X8-X9 is selected from the group consisting of: LA, LL, AL, AA, LC, LV, QA, KA, LS, LI, LY, and RA.
8 . The fusion protein according to claim 1 , wherein said protein expression enhancing polypeptide domain (1) comprises one of the following amino acid sequences:
Amino Acid Sequence
Sequence Identifier
123456789012345
SEQ ID NO: 48
IKKAYRKLA
SEQ ID NO: 49
IKKAYKLALQ
SEQ ID NO: 50
IKKAYRLALQ
SEQ ID NO: 51
IKKAYRKALQ
SEQ ID NO: 52
IKKAYRKLLQ
SEQ ID NO: 53
IKKYRKLA
SEQ ID NO: 54
IKKAYKLA
SEQ ID NO: 55
IKKAYRLA
SEQ ID NO: 56
IKKAYRKA
SEQ ID NO: 57
LKKAYRKLA
SEQ ID NO: 58
VKKAYRKLA
SEQ ID NO: 59
MKKAYRKLA
SEQ ID NO: 60
AKKAYRKLA
SEQ ID NO: 61
IAKAYRKLA
SEQ ID NO: 62
IKAAYRKLA
SEQ ID NO: 63
IKKRYRKLA
SEQ ID NO: 64
IKKSYRKLA
SEQ ID NO: 65
IKKQYRKLA
SEQ ID NO: 66
IKKEYRKLA
SEQ ID NO: 67
IKKFYRKLA
SEQ ID NO: 68
IKKCYRKLA
SEQ ID NO: 69
IKKAFRKLA
SEQ ID NO: 70
IKKAWRKLA
SEQ ID NO: 71
IKKAYRKQA
SEQ ID NO: 72
IKKAYRKMA
SEQ ID NO: 73
IKKAYRKIA
SEQ ID NO: 74
IKKAYRKAA
SEQ ID NO: 75
IKKAYRKVA
SEQ ID NO: 76
IKKAYRKRA
SEQ ID NO: 77
IKKAYRKLM
SEQ ID NO: 78
IKKAYRKLI
SEQ ID NO: 79
IKKAYRKLV
SEQ ID NO: 80
IKKAYRKLC
SEQ ID NO: 81
IKKAYRKLS
SEQ ID NO: 82
IKKAYRKLY
SEQ ID NO: 83
IRKAYRKLSLTL
SEQ ID NO: 84
IKKQYRLLSLKY
SEQ ID NO: 85
IKKAFHKLAMKY
SEQ ID NO: 86
IRQAFKKLALKL
SEQ ID NO: 87
IIKAYRKLALQW
SEQ ID NO: 88
IARAYRQLARRY
SEQ ID NO: 89
IKRAYRRQALRY
SEQ ID NO: 90
IKKSYRKLALKY
SEQ ID NO: 91
IKKAYKRLAMKY
9 . The fusion protein according to claim 1 , wherein the target protein of interest (2) is selected from the group consisting of: a soluble protein, a membrane-associated protein, a secreted protein, an extracellular domain of a transmembrane protein, an extracellular binding domain of a cell surface receptor fused to an Fc domain, an antibody or antigen-binding fragment thereof, a cytokine, a peptide hormone, an enzyme, and a morphogenetic protein.
10 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein according to claim 1 .
11 . A nucleic acid vector molecule comprising the isolated nucleic acid molecule according to claim 10 .
12 . A host cell comprising the nucleic acid vector molecule according to claim 11 .
13 . A method of expressing a fusion protein comprising (1) a protein expression enhancing polypeptide (PEEP) domain linked to (2) a target protein of interest comprising culturing a host cell according to claim 12 under conditions sufficient to produce the fusion protein.
14 . A method of expressing a fusion protein comprising (1) a protein expression enhancing polypeptide (PEEP) domain linked to (2) a target protein of interest comprising transfecting a host cell with an expression vector comprising a structural gene encoding a fusion protein according to claim 1 and culturing said transfected host cell under conditions causing the expression said fusion protein.
15 . A method of expressing a fusion protein comprising (1) a protein expression enhancing polypeptide (PEEP) domain linked to (2) a target protein of interest (2) comprising the steps of:
1) constructing a recombinant gene sequence encoding a fusion protein according to claim 1 ; 2) inserting the recombinant gene sequence into an expression vector to form a recombinant expression vector wherein said recombinant gene sequence is operably linked to a transcriptional promoter sequence; 3) transfecting said recombinant expression vector into host cells that are compatible with said promoter sequence; and 4) culturing said transfected host cells under conditions that permit expression of said fusion protein.
16 . A method of restoring a protein function in cells of a mammalian subject that are deficient in the secretion of a native secreted protein that provides said protein function comprising inserting into cells of the subject an exogenous nucleic acid molecule encoding a fusion protein according to claim 1 comprising (1) a protein expression enhancing polypeptide (PEEP) domain linked to (2) a target protein of interest, wherein said target protein of interest is the secreted protein, wherein expression of the fusion protein provides the function of the native secreted protein whose secretion is deficient in cells of the subject in the absence of the exogenous nucleic acid.
17 . The method according to claim 16 , wherein the subject has a disease associated with the deficient secretion of a native secreted protein in the subject.
18 . The method according to claim 17 , wherein said disease is selected from the group consisting of a prion-associated disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, cystic fibrosis (CF), and al-antitrypsin (AAT) deficiency.
19 . The method according to claim 18 , wherein the subject is a human subject deficient in the secretion of cystic fibrosis transmembrane conductance regulator protein and the disease is cystic fibrosis.
20 . An isolated protein expression enhancing polypeptide selected from the group consisting of:
(a) an active fragment of a J domain of a J protein; and (b) a J domain analog polypeptide comprising the amino acid sequence of formula I:
(I) X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO:47), wherein:
X1 is isoleucine (I), leucine (L), valine (V), alanine (A), or methionine (M);
X2 and X3 are each independently any amino acid with the proviso that one or both are K or R;
X4 is any amino acid or X4 may be absent when X1 through X3 are present and X5 through X9 are present;
X5 is tyrosine (Y), tryptophan (W), or phenylalanine (F);
X6 and X7 are each independently any amino acid with the proviso that one or both are lysine (K) or arginine (R); or either one of X6 and X7 may be absent when the other is K or R and when X1 through X5 are present and X8 and X9 are present; and
X8 and X9 are any amino acid with the proviso that one or both are leucine (L) or alanine (A); or one of X8 and X9 may be absent when the other is L or A and when X1 through X7 are present and wherein said J domain analog polypeptide is smaller than a J domain of a J protein; and
wherein a fusion protein comprising said protein expression enhancing polypeptide linked to a target protein of interest is expressed in a host cell at an enhanced level as compared to the level of expression of the unmodified target protein of interest; or wherein co-expression in a host cell of a target protein of interest and a fusion protein comprising said protein expression enhancing polypeptide linked to a target protein binding domain that binds said target protein of interset results in an enhanced level of expression of said target protein of interest as compared to the level of expression of said target protein of interest in the absence of said fusion protein.
21 . The protein expression enhancing polypeptide according to claim 20 , wherein said protein expression enhancing polypeptide is an active fragment of an isolated J domain of a J protein, wherein said J domain has an amino acid sequence selected from any one of SEQ ID NOs:1-46.
22 . The protein expression enhancing polypeptide according to claim 20 , wherein said protein expression enhancing polypeptide has any amino acid sequence selected from any one of SEQ ID NOs: 48-91.
23 . The protein expression enhancing polypeotide according to claim 1 , wherein the protein expression enhancing polypeptide is an active fragment of an isolated J domain of an Erdj protein selected from the group consisting of: Erdj1, Erdj2, Erdj3, Erdj4, Erdj5, Erdj6, and Erdj7.
24 . The protein expression enhancing fusion protein according to claim 23 , wherein the protein expression enhancing polypeptide is a fragment of an isolated J domain of Erdj3.
25 . The protein expression enhancing polypeptide according to claim 1 , wherein the protein expression enhancing polypeptide is a J domain analog polypeptide that comprises the amino acid sequence of formula I.
26 . The protein expression enhancing polypeptide according to claim 25 , wherein said J domain analog polypeptide comprises the amino acid sequence (SEQ ID NO:326) wherein:
X1 is I, L, V, A, or M; the dipeptide X2-X3 is selected from the group consisting of: KR, KK, RK, RR, AK, AR, KA, IK, NK, KQ, RQ, and RD; X4 is A, S, T, R, S, Q, E, F, C, or I; X5 is Y or F; the dipeptide X6-X7 is selected from the group consisting of: KR, KK, RK, RR, RQ, FR, RL, KL, HK, LK, QK, and KV; and the dipeptide X8-X9 is selected from the group consisting of: LA, LL, AL, AA, LC, LV, QA, KA, LS, LI, LY, and RA.
27 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a protein expression enhancing polypeptide according to claim 20 .
28 . A nucleic acid vector molecule comprising the isolated nucleic acid molecule according to claim 27 .
29 . A host cell comprising the nucleic acid vector molecule according to claim 28 .Join the waitlist — get patent alerts
Track US2018087081A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.