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KMID : 0603820080140020119
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Journal of Experimental & Biomedical Science
2008 Volume.14 No. 2 p.119 ~ p.122
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Functional Assessments of Spodpotera Cell-expressed Human Erythrocyte-type Glucose Transport Protein with a Site-directed Mutagenesis
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Lee Chong-Kee
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Abstract
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The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. In order to exploit this, the effects of substitution at the highly conserved residue glutamine 282 of the human erythrocyte-type glucose transporter have been examined by in vitro site-directed mutagenesis. The modified human transport protein has been expressed in Spodoptera frugiperda 21 cells by using the recombinant baculovirus AcNPV-GTL. To assess the functional integrity of the expressed transporter, measurements of the transport inhibitor cytochalasin B binding were performed, involving the membranes prepared from 4 days post infection with no virus, with wild-type virus or AcNPV-GTL virus. Data obtained showed that there was little or no D-glucose-inhibitable binding in cells infected with the wild type or no virus. Only the recombinant virus infected cells exhibited specific binding, which is inhibitable by D- but not by L-glucose. However, there was a notable reduction in the affinity for the potent inhibitor cytochalasin B when binding measurements of AcNPV-GTL were compared with those of AcNPV-GT, which has no substitution. It is thus suggested that although the modified and unmodified human transporters differed slightly in their affinity for cytochalasin B, the glutamine substitution did not interfere the heterologous expression of the human transporter in the insect cells.
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KEYWORD
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Baculovirus, Sf21 cells, Mutagenesis
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