SYSTEMS AND METHODS FOR EXPRESSING PROTEINS
The present disclosure relates to method and compositions for generating proteins. In particular, the present disclosure relates to electroporation mediated gene delivery in the generation of recombinant proteins (e.g., drug metabolizing enzyme and transporter vesicles) in mammalian cells.
1 . A method of generating a vesicle that expresses a membrane bound protein, comprising:
a) contacting a mammalian cell with a nucleic acid encoding a membrane bound protein selected from the group consisting of: ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCG2, and a homolog thereof;
b) electroporating said mammalian cell such that said nucleic acid enters said mammalian cell; and
c) isolating vesicles expressing said membrane bound protein of interest.
2 . The method of claim 1 , further comprising the step of culturing said cells after said electroporation step.
3 . The method of claim 2 , wherein said culturing comprises addition of sodium butyrate.
4 . The method of claim 1 , wherein said isolating step comprises homogenization of said cells.
5 . The method of claim 1 , wherein said membrane bound protein comprises post-translational modifications similar to the membrane bound protein in native form.
6 . The method of claim 1 , wherein said mammalian cell is a HEK293, CHO, Hela, S2, MDCK-I, MDCK-II, LLC-PK1, Caco-2, Huh7, or V79 cell.
7 . The method of claim 1 , further comprising the step of contacting said vesicle with a test compound.
8 . The method of claim 7 , wherein said test compound is a drug.
9 . A method of screening a test compound, comprising:
a) contacting a mammalian cell with a nucleic acid encoding a transporter protein selected from the group consisting of ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCG2, and a homolog thereof;
b) electroporating said mammalian cell such that said nucleic acid enters said mammalian cell;
c) isolating cell membranes expressing said transporter protein; and
d) contacting said cell membranes with said test compound.
10 . The method of claim 9 , further comprising the step of measuring transport of said test compound by said transporter protein.
11 . The method of claim 10 , wherein said measuring comprises measuring kinetics of transport.
12 . The method of claim 9 , further comprising the step of contacting said cell membrane with an inhibitor of said drug transporters and measuring inhibition of activity or transport of said drug transporters by said inhibitor.
13 . An isolated vesicle produced by the method of claim 9 .
14 . A method of generating a cell fraction containing a protein of interest, comprising:
a) contacting a mammalian cell with a nucleic acid encoding a drug metabolizing enzyme selected from the group consisting of a cytochromes P450, an aldehyde oxidase (AO), a flavin monooxygenase (FMO), a monoamine oxidase A and B (MAO A and B), an esterase, a N-acetyltransferase (NAT), a sulfotransferase (SULT), a uridine 5′-diphospho-glucuronosyltransferase (UGT), and a homolog thereof;
b) electroporating said mammalian cell such that said nucleic acid enters said mammalian cell; and
c) isolating a cell fraction containing said drug metabolizing enzyme.
15 . The method of claim 14 , wherein said cytochrome P450 is selected from the group consisting of CYP1A1, CYP1B1, CYP2A6, CYP2B6, CYP1A2, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A5, CYP3A7, CYP3A4, CYP4F2, and CYP2J2.
16 . The method of claim 14 , wherein said esterase is selected from the group consisting of carboxylesterase 1 (CES1), carboxylesterase 2 (CES2), paraoxonase 1 (PON1), carboxymethylenebutenolidase (CMBL), butyrylcholinesterase (BChE), arylacetamide deacetylase (AADAC), and alkaline phosphatase (AP).
17 . The method of claim 14 , wherein said UGT is selected from the group consisting of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2A1, UGT2A2, UGT2A3, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28.
18 . The method of claim 14 , further comprising the step of culturing said cells after said electroporation step.
19 . The method of claim 18 , where said culturing comprises addition of sodium butyrate.
20 - 24 . (canceled)
25 . The method of claim 14 , further comprising the step of contacting said cell fraction with a test compound.
26 . The method of claim 25 , wherein said test compound is a drug.
27 . (canceled)
28 . A cell fraction produced by the method of claim 14 .
29 . An isolated vesicle produced by the method of claim 1 .
30 . The method of claim 9 , further comprising the step of measuring transport of said test compound by said transporter protein.