Apotransferrin is internalized and distributed in the same way as holotransferrin in K562 cells.

Transferrin (Tf), a naturally existing protein, has received considerable attention in the area of drug targeting since it is biodegradable, non-toxic, and non-immunogenic. The efficient cellular uptake of Tf shows it has potential in the delivery of anti-cancer drugs, proteins, and therapeutic genes into proliferating malignant cells that overexpress transferrin receptor (TfR). In human serum, about 30% of Tf exists in the iron-saturated form (Fe(2)-Tf) and the remainder exists as apotransferrin (apo-Tf). Understanding the uptake of apo-Tf by cells will provide key insights into studies on Tf-mediated drug delivery. In the present study, we investigated visually the transport of apo-Tf into K562 cells and its intracellular localization by laser-scanning confocal microscopy (LSCM) and flow cytometry analysis (FCA). It was found that, like Fe(2)-Tf, apo-Tf can be taken up into the cells. The process is time- and temperature-dependent, competitively inhibited by Fe(2)-Tf, and significantly abolished by pronase pretreatment. Visual evidence showed that the transport of apo-Tf into K562 cells is a TfR-mediated process. Furthermore, the investigations using optical-slicing technique demonstrated that the distribution of apo-Tf is similar to that of Fe(2)-Tf, both appearing in the perinuclear region in ball-in-bowl shape.

Optimization of the expression of human DNA topoisomerase I in Pichia pastoris / 生物工程学报

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.