Systems and methods for expressing proteins

Abstract

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.

What is claimed is: 
     
         1 . 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.   
     
     
         2 . The method of  claim 1 , further comprising the step of measuring transport of said test compound by said transporter protein. 
     
     
         3 . The method of  claim 2 , wherein said measuring comprises measuring kinetics of transport. 
     
     
         4 . The method of  claim 1 , 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. 
     
     
         5 . 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.   
     
     
         6 . The method of  claim 5 , 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. 
     
     
         7 . The method of  claim 5 , wherein said esterase is selected from the group consisting of carboxylesterase 1 (CES1), carboxylesterase 2 (CES2), paraoxonase 1 (POND, carboxymethylenebutenolidase (CMBL), butyrylcholinesterase (BChE), arylacetamide deacetylase (AADAC), and alkaline phosphatase (AP). 
     
     
         8 . The method of  claim 5 , wherein said UGT is selected from the group consisting of UGT1A1, UGTIA3, UGT1A4, UGTIA5, UGT1A6, UGTIA7, UGT1A8, UGT1A9, UGT1A10, UGT2A1, UGT2A2, UGT2A3, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28. 
     
     
         9 . The method of  claim 5 , further comprising the step of culturing said cells after said electroporation step. 
     
     
         10 . The method of  claim 9 , where said culturing comprises addition of sodium butyrate. 
     
     
         11 . The method of  claim 5 , wherein said isolating step comprises homogenization of said cells. 
     
     
         12 . The method of  claim 5 , wherein the drug metabolizing enzyme comprises post-translational modifications similar to the drug metabolizing enzyme in native form. 
     
     
         13 . The method of  claim 5 , wherein said mammalian cell is HEK293, CHO, Hela, S2, MDCK-I, MDCK-II, LLC-PK1, Caco-2, Huh7, or V79 cell. 
     
     
         14 . The method of  claim 5 , wherein said cell fraction is a cytosolic fraction. 
     
     
         15 . The method of  claim 5 , wherein said cell fraction is a membrane fraction. 
     
     
         16 . The method of  claim 5 , further comprising the step of contacting said cell fraction with a test compound. 
     
     
         17 . The method of  claim 16 , wherein said test compound is a drug. 
     
     
         18 . The method of  claim 17 , further comprising the step of assessing modification of said drug. 
     
     
         19 . A cell fraction produced by the method of  claim 5 .