title: Effect of inducible co-overexpression of protein disulfide isomerase and endoplasmic reticulum oxidoreductase on the specific antibody productivity of recombinant Chinese hamster ovary cells.
abstract: To enhance specific antibody (Ab) productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells, post-translational limitations in the endoplasmic reticulum during antibody production should be relieved. Previously, we reported that overexpression of protein disulfide isomerase (PDI), which catalyzes disulfide bond exchanges and assists in protein folding of newly synthesized proteins, enhanced q(Ab) of rCHO cells by about 27% (Mohan et al., 2007, Biotechnol Bioeng 98:611-615) . Since the rate limiting step in disulfide bond formation is found to be the regeneration of oxidized PDI, the oxidation state of PDI, as well as the amount of PDI, might be important. Endoplasmic reticulum oxidoreductase (ERO1L) maintains PDI in an oxidized state so that disulfide bond formation occurs. Here, PDI and its helper protein, ERO1L were overexpressed in rCHO cells producing an Ab in an attempt to ease the bottleneck in disulfide bond formation, and hence, Ab folding and secretion. Transient expression of ERO1L alone and with PDI resulted in enhanced q(Ab) by 37% and 55%, respectively. In contrast, under stable inducible co-overexpression of PDI and ERO1L, the q(Ab) was unaffected or negatively affected by varying degrees, depending on the individual expression levels of these genes. In stable clones with altered oxidation state of PDI due to co-overexpression of PDI and ERO1L, secretion of Ab was hindered and PDI-associated retention of Ab was seen in the cells. Under transient gene expression, secretion of Ab was not compromised. The data presented here suggests a possible mechanism of PDI/ERO1L interaction with the target Ab and shows how the expression levels of these proteins could affect the q(Ab) of this Ab-producing rCHO cell line.
authors.affiliation: Department of Biological Sciences, Graduate School of Nanoscience & Technology, KAIST, Kusong-Dong, Yusong-Gu, South Korea.
authors.forename: Chaya
authors.initials: C
authors.lastname: Mohan
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D015870:Gene Expression; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis*; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000906:Antibodies; D004220:Disulfides; D011994:Recombinant Proteins; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases