PROTEIN EXPRESSION ENHANCING POLYPEPTIDES

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 .