Plasmid vectors with a 5'-hybrid intron facilitate high-level glycoprotein expression in CHO-cells.
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.
Melcher
Ralph
R
Department of Medicine, University of Wuerzburg, Gastrolabor/Bau 4, Joseph-Schneider-Str. 2, 97080 Wuerzburg, Germany. r.melcher@medizin.uni-wuerzburg.de
Grosch
Hans-Wilhelm
HW
Hasilik
Andrej
A
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D005822:Genetic Vectors; D007438:Introns / Q000235:genetics*; D009113:Muramidase / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D020121:5' Untranslated Regions; D011994:Recombinant Proteins; D009113:Muramidase