Date Swapneeta S, Fiori Mariana C, Altenberg Guillermo A, Jansen Michaela
Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, United States of America.
Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, United States of America.
PLoS One. 2017 May 11;12(5):e0177572. doi: 10.1371/journal.pone.0177572. eCollection 2017.
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.
质子偶联叶酸转运体（PCFT）为哺乳动物体内的维生素叶酸（B9）提供了一条重要的摄取途径。此外，由于快速分裂的肿瘤细胞对叶酸的需求增加，以及几种肿瘤中PCFT表达上调，目前它在将化疗药物靶向肿瘤方面备受关注。为了解其功能，确定其原子结构和转运分子机制是必不可少的目标，这需要大量有功能的PCFT。在此，我们利用重组杆状病毒和草地贪夜蛾（Sf9）昆虫细胞，展示了一种用于人PCFT的高效异源表达系统。我们在PCFT的表达、分离和纯化过程中证明了叶酸转运功能。重要的是，纯化后的PCFT在重构后能够转运叶酸。因此，我们成功克服了异源表达这一PCFT研究的主要瓶颈。人PCFT过表达系统的可用性为未来的生化、生物物理和结构研究奠定了基础。
