Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1)

Date, Swapneeta; Fiori, Mariana C.; Altenberg, Guillermo A.; Jansen, Michaela

doi:10.1371/journal.pone.0177572

Cited by 11 publications

(9 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The presentation by M. Jansen on the “Path to structural understanding of folate transport through PCFT” was aimed to understand PCFT function and determination of its atomic structure and molecular mechanism of transport. These approaches require large amounts of functional protein and several high-level heterologous expression systems were used for human PCFT including insect cells and yeast [ 14 ]. PCFT was functional in whole cell assays in both expression systems and after solubilization, purification to homogeneity, and reconstitution into liposomes.…”

Section: Structure and Biology Of The One-carbon Solute Transportersmentioning

confidence: 99%

Folate receptors and transporters: biological role and diagnostic/therapeutic targets in cancer and other diseases

Frigerio

Bizzoni

Jansen

et al. 2019

J Exp Clin Cancer Res

View full text Add to dashboard Cite

Folate receptors and transporters and one-carbon metabolism continue to be important areas of study given their essential roles in an assortment of diseases and as targets for treatment of cancer and inflammation. Reflecting this, every 2 years, the Folate Receptor Society organizes an international meeting, alternating between North America and Europe, where basic and translational scientists, clinical oncologists and rheumatologists from both academia and industry convene in an informal setting. The 7th International Symposium on Folate Receptors and Transporters was held in Sant’Alessio Siculo (ME), Taormina, Italy from 1st to 5th of October 2018, organized by Dr. Mariangela Figini from Fondazione IRCCS Istituto Nazionale dei Tumori, Milan. Following the format of previous meetings, more than 50 scientists from 9 different countries attended the 2018 meeting to share ongoing developments, discuss current research challenges and identify new avenues in basic and translational research. An important feature of this meeting was the participation of young investigators and trainees in this area, two (A. Dekhne and N. Verweij) of whom were awarded fellowships to attend this meeting as a recognition of the high scientific quality of their work. This report provides a synopsis of the highlights presented in the following sessions: Barton Kamen Lecture; Targeting one-carbon metabolism in cytosol and mitochondria; Structure and biology of the one-carbon solute transporters; Physiology and pathophysiology of folate receptors and transporters; Folate receptors for targeting tumors and inflammatory diseases; Conventional and new anti-folate drugs for treating inflammatory diseases and cancer; Imaging; Ongoing clinical trials; and Chimeric Antigen Receptor cell therapies of cancer.

show abstract

Section: Structure and Biology Of The One-carbon Solute Transportersmentioning

confidence: 99%

Folate receptors and transporters: biological role and diagnostic/therapeutic targets in cancer and other diseases

Frigerio

Bizzoni

Jansen

et al. 2019

J Exp Clin Cancer Res

View full text Add to dashboard Cite

show abstract

“…With the increasing demand for recombinant proteins, and continued discovery of novel medically useful proteins, the improvement of production technology has become a focus of research exploration. Various excellent expression systems based on different hosts have gradually become the primary tools for protein production, including plant [ 1 ], Escherichia coli ( E. coli ) [ 2 ], insect [ 3 ], yeast [ 4 ], and mammals [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

Ruan

et al. 2021

IJMS

View full text Add to dashboard Cite

Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm’s genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.

show abstract

“…PCFT was identified as the main pathway for dietary folate uptake in the proximal small intestine in 2006 [ 5 ]. Detailed studies of human PCFT using PCFT constructs modified with amino acids engineered at specific positions, including the use of the substituted cysteine accessibly method (SCAM) [ 57 ] as well as recent cryo-EM structures of chicken PCFT have provided insights into the topology and conformational transitions during substrate translocation [ 15 , 17 , 33 – 40 , 45 , 58 – 61 ]. However, much remains unknown about PCFT’s detailed molecular structure, substrate translocation mechanism, and lipid dependence.…”

Section: Discussionmentioning

confidence: 99%

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter

et al. 2021

Self Cite

View full text Add to dashboard Cite

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

show abstract

Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1)

Cited by 11 publications

References 51 publications

Folate receptors and transporters: biological role and diagnostic/therapeutic targets in cancer and other diseases

Folate receptors and transporters: biological role and diagnostic/therapeutic targets in cancer and other diseases

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter

Contact Info

Product

Resources

About