Cell fraction and method of generating a cell fraction containing a protein of interest
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 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;
c) culturing said cell, where said culturing comprises addition of sodium butyrate; and
d) isolating a cell fraction containing said drug metabolizing enzyme, thereby generating the cell fraction containing the protein of interest,
wherein the generated cell fraction expressing the said metabolizing enzyme has more activity per mg protein than a baculovirus expression system cell fraction.
2. The method of claim 1 , 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.
3. The method of claim 1 , 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).
4. The method of claim 1 , 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.
5. The method of claim 1 , wherein said isolating step comprises homogenization of said cells.
6. The method of claim 1 , wherein the drug metabolizing enzyme comprises post-translational modifications similar to the drug metabolizing enzyme in native form.
7. The method of claim 1 , wherein said mammalian cell is HEK293, CHO, Hela, S2, MDCK-I, MDCK-II, LLC-PK1, Caco-2, Huh7, or V79 cell.
8. The method of claim 1 , wherein said cell fraction is a cytosolic fraction.
9. The method of claim 1 , wherein said cell fraction is a membrane fraction.
10. The method of claim 1 , further comprising the step of contacting said cell fraction with a test compound.
11. The method of claim 10 , wherein said test compound is a drug.
12. The method of claim 11 , further comprising the step of assessing modification of said drug.
13. A cell fraction produced by the method of claim 1 .