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.
Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, beta-galactosidase or N-Acetyl-beta-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions.
The different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sialylated forms by comparison to the control subjects. Psoriasis patients who had no evidence of alcohol abuse had similar profile for the isoforms of transferrin to that of the controls. Other groups of patients with alcohol-induced tissue damage, i.e. liver, brain or muscle, used as positive controls, similarly showed significant increases in the 2-sialylated forms, by comparison to controls. These results substantiate the current use of carbohydrate-deficient transferrin as a sensitive marker of alcohol abuse, particularly in subjects not drinking in excess of 60 g of ethanol/day but showing alcohol-related psoriasis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.