Increasing productivity of arylsulfatase B-producing cell line by coexpression of formylglycine-generating enzyme

Abstract

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.

Keywords

• cho cell line
• arylsulfatase b
• galsulfase
• arsb gene
• lysosomal enzyme
• sulfatase
• mucopolysaccharidosis type vi
• formylglycine-generating enzyme