Vectors for Recombinant Protein Expression in E. Coli
The present invention relates to methods of providing a protein product to a customer. In particular, the invention relates methods of using protein expression vectors to produce proteins to be provided to a client. The invention also provides vectors for the cloning and expression of proteins, including reagent proteins and therapeutic proteins.
1 . A method of providing a therapeutic protein to a customer, said method comprising cloning a nucleic acid encoding said protein into a pCWinl expression vector as set forth in SEQ ID NO:1, expressing said protein therefrom, and providing said protein to said customer.
2 . A method of providing a therapeutic protein to a customer, said method comprising cloning a nucleic acid encoding said protein into a pCWin 2 expression vector as set forth in SEQ ID NO:2, expressing said protein therefrom, and providing said protein to said customer.
3 . A method of providing a therapeutic protein to a customer, said method comprising cloning a nucleic acid encoding said protein into a nucleic acid vector selected from the group consisting of:
a) a pCWin2/MBP expression vector as set forth in SEQ ID NO:3;
b) a pCWin2-MBP-SBD (pMS 39 ) expression vector as set forth in SEQ ID NO:10; and
c) a pCWin2-MBP-MCS-SBD (pMXSp 39 ) expression vector as set forth in SEQ ID NO:11;
expressing said protein therefrom, and providing said protein to said customer.
4 . The method of claim 3 , wherein said nucleic acid vector comprises a protease cleavage site coding sequence at a location selected from the group consisting of:
a) between the MBP coding sequence and the therapeutic protein coding sequence; and
b) immediately prior to the start of the C-terminus of the MBP coding sequence.
5 . The method of claim 2 or 3 , wherein said protein is selected from the group consisting of erythropoietin, human growth hormone, granulocyte colony stimulating factor, interferons alpha, -beta, and -gamma, Factor IX, follicle stimulating hormone, interleukin-2, erythropoietin, anti-TNF-alpha, and a lysosomal hydrolase.
6 . The method of claim 5 , wherein said lysosomal hydrolase is selected from the group consisting of beta-glucosidase, alpha-galactosidase-A, beta-hexosaminidase, beta-galactosidase, alpha-galactosidase, alpha-mannosidase, beta-mannosidase, alpha-L-fucosidase, beta-glucuronidase, alpha-glucosidase, alpha-N-acetylgalactosaminidase, and acid phosphatase.
7 . A method of providing a protein to a customer, said method comprising cloning a nucleic acid encoding said protein into a pCWin1 expression vector as set forth in SEQ ID NO:1, expressing said protein therefrom, and providing said protein to said customer.
8 . A method of providing a protein to a customer, said method comprising cloning a nucleic acid encoding said protein into a pCWin2 expression vector as set forth in SEQ ID NO:2, expressing said protein therefrom, and providing said protein to said customer.
9 . A method of providing a protein to a customer, said method comprising cloning a nucleic acid encoding said protein into a nucleic acid vector selected from the group consisting of:
a) a pCWin2/MBP expression vector as set forth in SEQ ID NO:3;
b) a pCWin2-MBP-SBD (pMS 39 ) expression vector as set forth in SEQ ID NO:10; and
c) a pCWin2-MBP-MCS-SBD (pMXS 39 ) expression vector as set forth in SEQ ID NO:11;
expressing said protein therefrom, and providing said protein to said customer.
10 . The method of claim 7 , 8 or 9 , wherein said protein is selected from the group consisting of a glycosyltransferase and a sugar nucleotide-generating enzyme.
11 . A method of providing a protein to a customer, said method comprising providing a pCWin1 vector as set forth in SEQ ID NO:1 to a protein production facility, wherein a nucleic acid encoding said protein is cloned into said expression vector and said protein is expressed therefrom in said protein production facility, and providing said protein to said customer.
12 . A method of providing a protein to a customer, said method comprising providing a pCWin2 vector as set forth in SEQ ID NO:2 to a protein production facility, wherein a nucleic acid encoding said protein is cloned into said expression vector and said protein is expressed therefrom in said protein production facility, and providing said protein to said customer.
13 . A method of providing a protein to a customer, said method comprising providing a nucleic acid vector selected from the group consisting of:
a) a pCWin2/MBP expression vector as set forth in SEQ ID NO:3;
b) a pCWin2-MBP-SBD (pMS 39 ) expression vector as set forth in SEQ ID NO:10; and
c) a pCWin2-MBP-MCS-SBD (pMXS 39 ) expression vector as set forth in SEQ ID NO:11;
to a protein production facility, wherein a nucleic acid encoding said protein is cloned into said expression vector and said protein is expressed therefrom in said protein production facility, and providing said protein to said customer.
14 . The method of claim 2 , 3 , 4 , 7 , 8 or 9 , wherein said method further comprises prior to providing said protein to said customer, at least one glycosyl moiety is added to said protein.
15 . The method of claim 14 , wherein said glycosyl moiety is added to said protein in vitro.
16 . A method of providing a protein to a customer, said method comprising cloning a nucleic acid encoding said protein into-nucleic acid vector selected from the group consisting of:
a) a pCWin1 vector as set forth in SEQ ID NO:1;
b) a pCWin2 vector as set forth in SEQ ID NO:2;
c) a pCWin2/MBP vector as set forth in SEQ ID NO:3;
d) a pCWin2-MBP-SBD (pMS 39 ) vector as set forth in SEQ ID NO:10; and
e) a pCWin2-MBP-MCS-SBD (pMXS 39 ) vector as set forth in SEQ ID NO:11;
further wherein said method comprises inserting said vector into a bacterial host cell, expressing said protein in said host cell, and providing said protein to said customer.
17 . The method of claim 16 , wherein said method further comprises prior to providing said protein to said customer, at least one glycosyl moiety is added to said protein.
18 . The method of claim 16 , wherein said glycosyl moiety is added to said protein in vitro.
19 . The method of claim 16 , wherein said expression vector further comprises an affinity tag coding sequence.
20 . An isolated pcWIN1 expression vector comprising the sequence set forth in SEQ ID NO:1.
21 . An isolated pcWIN1 expression vector consisting of the sequence set forth in SEQ ID NO:1.
22 . An isolated pcWIN2 expression vector comprising the sequence set forth in SEQ ID NO:2.
23 . An isolated pcWIN2 expression vector consisting of the sequence set forth in SEQ ID NO:2.
24 . An isolated pcWIN2/MBP expression vector comprising the sequence set forth in SEQ ID NO:3.
25 . An isolated pcWIN2/MBP expression vector consisting of the sequence set forth in SEQ ID NO:3.
26 . The pcWIN280P expression vector of claim 24 , wherein the pCWIN2/MBP vector comprises a protease cleavage site coding sequence adjacent to the MBP coding sequence.
27 . An isolated pCWin2-MBP-SBD (pMS 39 ) vector comprising the sequence set forth in SEQ ID NO:10.
28 . An isolated pCWin2-MBP-SBD (pMS 39 ) vector consisting of the sequence set forth in SEQ ID NO:10.
29 . An isolated pCWin2-MBP-MCS-SBD (pMXS 39 ) vector comprising the sequence set forth in SEQ ID NO:11.
30 . An isolated pCWin2-MBP-MCS-SBD (pMXS 39 ) vector consisting of the sequence set forth in SEQ ID NO:11.
31 . The pCWin2-MBP-SBD (pMS 39 ) expression vector of claim 27 , wherein the pCWin2-MBP-SBD (pMS 39 ) vector comprises a protease cleavage site coding sequence immediately prior to the start of the C-terminus of the MBP coding sequence.
32 . A method of expressing a protein, said method comprising cloning a nucleic acid encoding said protein into a pCWin1 expression vector as set forth in SEQ ID NO:1 and expressing said protein therefrom.
33 . A method of expressing a protein, said method comprising cloning a nucleic acid encoding said protein into a pCWin2 expression vector as set forth in SEQ ID NO:2 and expressing said protein therefrom.
34 . A method of expressing a protein, said method comprising cloning a nucleic acid encoding said protein into a nucleic acid vector selected from the group consisting of:
a) a pCWin2/MBP expression vector as set forth in SEQ ID NO:3;
b) a pCWin2-MBP-SBD (pMS 39 ) expression vector as set forth in SEQ ID NO:10; and c) a pCWin2-MBP-MCS-SBD (pMXS 39 ) expression vector as set forth in SEQ ID NO:11;
and expressing said protein therefrom.
35 . The method of any one of claims 32 - 34 , wherein said protein is expressed in a prokaryotic cell.