Stable expression of recombinant human alpha3/4 fucosyltransferase III in Spodoptera frugiperda Sf9 cells.
Human alpha3/4 fucosyltransferase III (FT3; EC 2.4.1.65) synthesizes fucosylated glycoconjugates, namely the Lewis (Le) determinants. FT3 is detected in milk, gastric mucosa, kidney and other organs, but is found in very low amounts in these native tissues. In this work, we describe the expression of a soluble secretory form of FT3 (SFT3) in Spodoptera frugiperda (Sf9) insect cells using a non-lytic vector system. The coding sequence was cloned into the expression vector pIB/V5-His-TOPO which contains the transcriptional control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected cells were selected using blasticidin-HCl. It was observed that the secreted activity SFT3 increased until the sixth day of culture when it reached the value 1.9 mU x 10(-6) cells and 13.4 mg/l, whereas only 5% of activity was retained inside the cells. Western blot analysis of secreted and intracellularly retained SFT3 had a similar variation. Comparison of the stable with the lytic baculovirus expression system showed that the former yielded approx. 13-fold more active SFT3, which was possibly due to a lower accumulation of intracellular SFT3.
Morais
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VA
Instituto de Tecnologia Química e Biológica, Apartado 127, Avenida da República (EAN), Oeiras 2781-901, Portugal.
Costa
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D000818:Animals; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation; D004795:Enzyme Stability; D005647:Fucosyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase