Lupac Bifunctional Marker and Its Use in Protein Production
This invention relates to industrial production of proteins. More specifically, the invention relates to the Lupac surrogate marker, which corresponds to a fusion between luciferase and the puromycin N-acetyl transferase. The invention further relates to the use of Lupac for screening cells for high expression of a protein of interest.
1 - 38 . (canceled)
39 . A composition of matter comprising:
a) a Lupac polypeptide comprising a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
b) a Lupac polypeptide comprising a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
c) a Lupac polypeptide comprising a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
d) a Lupac polypeptide comprising a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
e) a Lupac polypeptide comprising a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase;
f) SEQ ID NO: 2;
g) a nucleic acid encoding a Lupac polypeptide comprising:
i) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
ii) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
iii) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
iv) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
v) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase; or
vi) SEQ ID NO: 2;
h) SEQ ID NO: 1;
i) a vector comprising:
i) SEQ ID NO: 1; or
ii) a nucleic acid encoding a Lupac polypeptide comprising:
A) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
B) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
C) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
D) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
E) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase; or
F) SEQ ID NO: 2; or
j) a host cell comprising a vector, said vector comprising:
i) SEQ ID NO: 1; or
ii) a nucleic acid encoding a Lupac polypeptide comprising:
A) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
B) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
C) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
D) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
E) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase; or
F) SEQ ID NO: 2.
40 . The composition of matter according to claim 39 , wherein said composition of matter is a vector and said vector further comprises a nucleic acid encoding a protein of interest.
41 . The composition of matter according to claim 40 , wherein said vector comprises at least two promoters, one driving the expression of said Lupac polypeptide and the other driving the expression of said protein of interest.
42 . The composition of matter according to claim 41 , wherein said at least two promoters are promoters of the murine CMV immediate early region.
43 . The composition of matter according to claim 42 , wherein said at least two promoters are the IE1 and the IE2 promoters.
44 . The composition of matter according to claim 39 , wherein said composition of matter is a vector and said vector further comprises an amplification marker selected from the group consisting of adenosine deaminase (ADA), dihydrofolate reductase (DHFR), multiple drug resistance gene (MDR), ornithine decarboxylase (ODC) and N-(phosphonacetyl) -L-aspartate resistance (CAD).
45 . The composition of matter according to claim 39 , wherein said composition of matter is a host cell and said host cell is a mammalian cell.
46 . The composition of matter according to claim 45 , wherein said mammalian cell is a CHO cell.
47 . The composition of matter according to claim 45 , wherein said mammalian cell is a human cell.
48 . A method of screening cells for expression of a protein of interest, said method comprising the step of:
(i) transfecting cells by the expression vector;
(ii) selecting cells being resistant to puromycin; and
(iii) assaying the luciferase activity of the cells selected in step (ii);
wherein said expression vector comprises a nucleic acid encoding a protein of interest and:
i) SEQ ID NO: 1; or
ii) a nucleic acid encoding a Lupac polypeptide comprising:
A) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
B) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
C) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
D) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
E) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase; or
F) SEQ ID NO: 2.
49 . The method according to claim 48 , wherein the 5%, 10%, 15% or 20% of cells that exhibit the highest luciferase activity in step (iii) comprise the cell that exhibit the highest expression of said protein of interest.
50 . The method according to claim 48 , wherein the luciferase activity of at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000 or 1,000,000 cells is assayed at step (iii).
51 . The method according to claim 49 , wherein the luciferase activity of at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000 or 1,000,000 cells is assayed at step (iii).
52 . The method according to claim 48 , further comprising the step of:
(iv) selecting about 1% to about 20% of the cells assayed in step (iii), wherein the selected cells are those exhibiting the highest luciferase activity in step (iii).
53 . The method according to claim 49 , further comprising the step of:
(iv) selecting about 1% to about 20% of the cells assayed in step (iii), wherein the selected cells are those exhibiting the highest luciferase activity in step (iii).
54 . The method according to claim 50 , further comprising the step of:
(iv) selecting about 1% to about 20% of the cells assayed in step (iii), wherein the selected cells are those exhibiting the highest luciferase activity in step (iii).
55 . The method according to claim 51 , further comprising the step of:
(iv) selecting about 1% to about 20% of the cells assayed in step (iii), wherein the selected cells are those exhibiting the highest luciferase activity in step (iii).
56 . The method according to claim 48 , further comprising the step of:
(v) assaying the expression level of the protein of interest in the cells selected at the end of step (iv).
57 . A method of obtaining a cell line expressing a protein of interest, said method comprising the step of:
(a) screening cells screening cells for expression of a protein of interest, said method comprising the step of:
(i) transfecting cells by the expression vector;
(ii) selecting cells being resistant to puromycin; and
(iii) assaying the luciferase activity of the cells selected in step (ii);
wherein said expression vector comprises a nucleic acid encoding a protein of interest and:
i) SEQ ID NO: 1; or
ii) a nucleic acid encoding a Lupac polypeptide comprising:
A) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
B) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
C) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
D) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
E) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase; or
F) SEQ ID NO: 2;
(b) selecting the cell exhibiting the highest expression of said protein of interest; and
(c) establishing a cell line from said cell.
58 . A method of producing a protein of interest, said method comprising the step of:
(a) culturing a cell line obtained according to the method according to claim 57 under conditions which permit expression of said protein of interest; and
(b) collecting said protein of interest.
59 . The method according to claim 58 , further comprising the step of purifying said protein of interest.
60 . The method according to claim 59 , further comprising the step of formulating said protein of interest into a pharmaceutical composition.
61 . A method of producing a Lupac polypeptide comprising:
(a) culturing a host cell comprising a vector, said vector comprising:
i) SEQ ID NO: 1; or
ii) a nucleic acid encoding a Lupac polypeptide comprising:
A) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity;
B) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8;
C) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac comprises amino acids 2 to 199 of SEQ ID NO: 9;
D) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a luciferase is fused to the 5′ terminus of said fragment of a pac;
E) a fragment of a luciferase fused to a fragment of a puromycin N-acetyl transferase (pac), wherein said Lupac polypeptide exhibits: (i) luciferase activity; and (ii) puromycin N-acetyl transferase activity, wherein said fragment of a pac is fused to the 5′ terminus of said fragment of a luciferase; or
F) SEQ ID NO: 2; and
(b) collecting the Lupac polypeptide produced by said host cell.
62 . The method according to claim 61 , further comprising the step of purifying said Lupac polypeptide.