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

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.

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

The THE INFLUENCE OF CHLORPROMAZINE HYDROCHLORIDE ON THE SIZE OF SMALL UNILAMELLAR DIMYROSTYLPHOSPHATIDYLCHOLINE LIPOSOMES AS REVEALED BY LIGHT SCATTERING PHOTOMETRY

Objectives: This study aims to investigate the possible influence of the model, cationic, surface-active solute chlorpromazine hydrochloride (CPZ-HCl) on the size of small unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes as a function of temperature and CPZ-HCl concentration, below and above the critical micelle concentration (CMC). Methods: Small unilamellar DMPC liposomes were prepared by dissolving DMPC in chloroform and the solvent was rota-evaporated in a water bath adjusted at 40 °C. The lipid film was then dispersed in 0.1 M KCl solution adjusted at pH 6.2 to form large multilamellar liposomes which are then sonicated and fractionated via Sepharose 2B-Cl gel. The elution profile was followed spectrophotometrically at λ 260 nm. Combined fractions from the trailing edge of the included peak which is due to small unilamellar liposomes, were used as a source throughout this study. The SOFICA light scattering photometer (Model 42000) was used to determine the weight average liposomes weight (Lw) of small unilamellar DMPC liposomes. The Lw was determined in the absence and presence of CPZ-HCl both above and below the CMC over the temperature range of 25 °C to 40 °C. Results: The Lw was observed to increase linearly in the absence and presence of CPZ-HCl. The Lw was observed to increase linearly in the absence of CPZ-HCl, from 1.88×106+0.02 g/mol at 25 °C to 3.25×106+0.03 g/mol at 40 °C. Similarly, the Lw was observed to increase linearly in the present of CPZ-HCl, for example at 18 mmol drug concentration, the Lw increases from 11×106+0.04 g/mol at 25 °C to 13.75×106+0.03 g/mol at 40 °C. When the data are presented as a function of CPZ-HCl concentration, a gradual increase in Lw was observed below the CMC. Little increase in Lw however, was observed at post-micellar concentrations of 14 mmol and 18 mmol.