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Expression and characterization of the human erythrocyte glucose transporter in insect cells using a recombinant baculovirus

Yi, Chong-Kee; (1994) Expression and characterization of the human erythrocyte glucose transporter in insect cells using a recombinant baculovirus. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The GLUT1 isoform of the mammalian passive glucose transporter family plays a physiologically vital role in catalysing the uptake of sugar by a variety of tissues, including human erythrocytes and the brain. Although the transporter can be purified from natural sources in small amounts, investigation of its structure and function, by techniques such as site-directed mutagenesis and by biophysical methods such as crystallization, would be greatly facilitated by development of heterologous expression systems capable of producing large amounts of the recombinant protein. To this end, a recombinant baculovirus was produced by inserting cDNA encoding human GLUT1 into the genome of the Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter. Insect cells infected with the recombinant virus were found to synthesise high levels of recombinant transporter (up to 8[percent] of total membrane protein). The expressed protein was recognised by a range of polyclonal, site-directed antibodies against GLUT1, indicating that it corresponded to a full-length version of GLUT1. However, its electrophoretic mobility on SDS gels was substantially greater than that of the natural erythrocyte protein, probably as a result of the recombinant protein being less heavily and heterogeneously glycosylated. Four days after infection with the virus, the insect cell-surface and intracellular membranes exhibited > 200 pmol cytochalasin B binding sites per mg protein, which bound the transport inhibitor with characteristics identical to those of the erythrocyte protein. The inhibitor also photolabelled the expressed protein in a D-glucose sensitive manner. Thus, it appears that expression of GLUT1 in insect cells yields a protein that is functionally similar to its erythrocyte counterpart. This is the first demonstration of overexpression of a mammalian facilitative glucose transporter in functional form. An attempt was also made to develop procedures for the rapid isolation of the recombinant transporter in functional form from insect cells, by producing a fusion protein containing a polyhistidine domain: the high affinity of the latter for metal ions should facilitate isolation of such fusion proteins by chromatography on columns of immobilised Ni2+. However, the low level of expression and apparent instability of the recombinant fusion protein encoding the transporter unfortunately precluded its purification by this approach.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Expression and characterization of the human erythrocyte glucose transporter in insect cells using a recombinant baculovirus
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10098595
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