High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay.

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with ＞95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

Betulin Suppresses Osteoclast Formation via Down-Regulating NFATc1.

Osteoporosis is a disease characterized by osteoclast-mediated low bone mass. The modulation of osteoclasts is important for the prevention or therapeutic treatment of loss of bone mass. Osteoclasts, which are bone-resorbing multinucleated cells, are differentiated from the hematopoietic stem cell monocyte/macrophage lineage by Receptor activator of nuclear factor kappa-B ligand (RANKL) expressed from osteoblasts and stromal cells. RANKL signaling ultimately activates nuclear factor of activated T Cells 1 (NFATc1), which is a master transcription factor in osteoclastogenesis. Betulin, a lupine type pentacyclic triterpenoid, was isolated from the bark of Betula platyphylla. Betulin inhibited RANKL-mediated osteoclast differentiation by downregulating NFATc1. Betulin may serve as a useful structural scaffold in the therapeutic agent development to prevention/treatment the osteoclast-mediated bone disorder.

Recombinant Glargine Insulin Production Process Using Escherichia coli.

Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into Escherichia coli JM109. The transformed E. coli cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.