Characterization of the human transferrin receptor produced in a baculovirus expression system.

D L Domingo,I S Trowbridge

doi:10.1016/s0021-9258(18)37716-0

Abstract

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.

Highlights

The transferrin receptor binds the serum transport glycoproteintransferrin and mediates iron uptakeinto the cell
We report the characterization of the human transferrin receptor produced in a baculovirus expression system [14, 15]
Cells not attached to the antibody-coated magnetic particles were Transferrin Receptor-An important step in the use of the removed, fresh media were added, and theseparation procedure was baculovirus expression system to produce recombinant prorepeated three tofive more times

Summary

Introduction

The transferrin receptor binds the serum transport glycoproteintransferrin and mediates iron uptakeinto the cell (reviewedin Refs. 1and 2). The structural requirements for the binding of transferrin to thereceptor, its localization in coated pits, endocytosis, and recycling back to the cell surface have not been defined. Each of these steps may require either specific protein-protein interactions, covalent modification of receptors, such as acylation or phosphorylation, conformational changes in receptor structure, or alterations in the stateof receptor aggregation, As with other cell surface receptors that either transduce signals or transport macromolecules, a full understanding of the structurefunction relationshipof the transferrin receptor is unlikely to be achieved without knowledge of its three-dimensional structure. An immunoselection technique employing antibody-coated magnetic particles that is generally applicable to other cell surface molecules was used to initially enrich for virus-infected cells producing the human transferrin receptor. B3/25 is a mouse anti-human transferrin receptor monoclonal antibody [11].Magnetic particles coated with goat anti-mouse IgG and Corning magnetic separator were fromAdvanced Magnetics, Inc., Cambridge, MA.

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