Plasmid vectors with a 5′-hybrid intron facilitate high-level glycoprotein expression in CHO-cells

• Published in: Biochimica et biophysica acta Vol. 1575; no. 1; pp. 49 - 53
• Main Authors: Melcher, Ralph, Grosch, Hans-Wilhelm, Hasilik, Andrej
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 03-05-2002
• Subjects: 5' Untranslated Regions - genetics; Animals; CHO Cells; CMV promoter; Cricetinae; Gene Expression; Genetic Vectors; Introns - genetics; Muramidase - biosynthesis; Muramidase - genetics; Plasmids - genetics; Promoter Regions, Genetic - physiology; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; SV-40 promoter; Tripartite leader sequence; CMV promoter; SV-40 promoter; Tripartite leader sequence
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(02)00242-7

For biosynthesis of recombinant glycoproteins with specified carbohydrate structures various Chinese hamster ovary (CHO) cell lines are available that express different sets of glycosyl transferases. To examine various forms of glycosylated lysozyme we prepared a vector that directs the synthesis of the recombinant glycoprotein at a high rate. We compared vectors with varied promoter and 5′-untranslated regions. The expression of cDNA of a glycosylated mutant lysozyme was examined under a control of the SV40 early and cytomegalovirus (CMV) promoters alone and in combination with a tripartite leader and a hybrid intervening sequence. We show that in this system a vector with the CMV promoter, the tripartite leader sequence and the intron, referred to as pMCI, is the best of the examined combinations. Using conventional tissue culturing of CHO cells stably transfected with this vector, we were able to isolate glycosylated lysozyme with a yield of 4.5 mg per liter of spent medium.