Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter PCFT, SLC46A1Report as inadecuate




Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter PCFT, SLC46A1 - Download this document for free, or read online. Document in PDF available to download.

The proton-coupled folate transporter PCFT provides an essential uptake route for the vitamin folic acid B9 in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda Sf9 insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.



Author: Swapneeta S. Date, Mariana C. Fiori, Guillermo A. Altenberg, Michaela Jansen

Source: http://plos.srce.hr/



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