RESUMO
Recombinant human transferrin as well as N- and C-terminal half-transferrins, produced in Escherichia coli, are deposited in inclusion bodies by the bacteria. The isolation and purification of the recombinant proteins from these inclusion bodies are described here. The amino acid compositions and N-terminal sequences of the proteins were determined, and found to be in agreement with the known protein structure of human serum transferrin. Renaturation of the recombinant proteins is described, resulting in water-soluble iron-binding molecules. Iron binding was confirmed by 59Fe labelling, absorption spectrophotometry and EPR spectrometry.
Assuntos
Transferrina/isolamento & purificação , Sequência de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transferrina/químicaRESUMO
1. Endosomes were isolated from K 562 cells after 3 min after the endocytosis of a single cohort of transferrin molecules. 2. The change in 125I/59Fe ratio of heavy and light endosomes, relative to that of the transferrin used and 59Fe from light endosomes. 3. Incubation of heavy and light endosomes with PBS or PIH showed equal ATP specific iron release from both heavy and light endosomes, but in the presence of a NADH/NAD+ redox couple iron release from light endosomes was reduced. 4. Incubation of heavy and light endosomes with PIH and NEM did not completely abolish ATP specific iron release from heavy and light endosomes.
