Development of novel galactosylation method for the expression of recombinant human transferrin in insect cell culture

• Auteur principal: Yap, Wei Ney
• Format: Thèse
• Langue: anglais
• Publié: 2006
• Sujets: QH301 Biology; TP Chemical technology
• Accès en ligne: http://eprints.utm.my/2145/1/YapWeiNeyMFKKSA2006.pdf

The objective of this research is to develop a novel galactosylation method for the expression of recombinant human transferrin (hTf) with better N-glycan quality. The baculovirus-insect cell system, consisting of hTf as the model protein, 1,4-galactosyltransferase (1,4-GalT) as the enzyme, and uridine-diphosphogalactose (UDP-Gal) as the sugar nucleotide, has been successfully established. In the early part of the study, fundamental works were carried out to optimize Spodoptera frugiperda (Sf-9) cells growth and mock infection. Serum concentration, different type of media, cell subculturing condition, initial cell density and spent medium carry over had been found to significantly influence the growth kinetics of Sf-9 cells. Multiplicity of infection (MOI) and spent medium carry over were found to have direct impact on viral infectivity. The optimized parameters were then used to evaluate the expression of recombinant hTf and 1,4-GalT in Sf-9 cells. Subsequently, native UDP-Gal levels at normal and upon baculovirus infection produced in Sf-9 cells were monitored using Reverse Phase High Performance Liquid Chromatography. UDP-Gal concentration was discovered to decrease gradually once infected with the recombinant baculovirus. Finally, baculovirus coinfection study was carried out to evaluate the recombinant glycoprotein quality. However, lectin binding analysis using Ricinus communis agglutinin-I, revealed that coexpression between rhTf and -1,4GalT (in vivo) did not show encouraging result due to the reduction of UDP-Gal upon baculovirus infection. This finding suggested that the introduction of -1,4GalT alone was not sufficient for successful galactosylation. However, another strategy was used to overcome the problem. Commercial GalT and UDP-Gal were introduced artificially to the rhTf after it was secreted from cell culture. It was found that the in vitro strategy promoted better Nglycan quality in insect cells