Effect of PDI overexpression on recombinant protein secretion in CHO cells.
In eukaryotic cells, protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. Increasing PDI activity in bacterial, yeast, and insect cell expression systems can lead to increased secretion of heterologous proteins containing disulfide bridges. Since Chinese hamster ovary (CHO) cells are widely used for the expression of recombinant proteins, we expressed recombinant human PDI (rhu PDI) in CHO cells to increase cellular PDI levels and examined its effect on the secretion of two different recombinant proteins: interleukin 15 (IL-15) and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). Secretion of TNFR:Fc (a disulfide-rich protein) is decreased in cells overexpressing PDI; the TNFR:Fc protein is retained inside these cells and colocalizes with the overexpressed rhu PDI protein in the endoplasmic reticulum. PDI overexpression did not result in intracellular retention of IL15. The nature of the interaction between PDI and TNFR:Fc was further investigated by expressing a disulfide isomerase mutant PDI in CHO cells to determine if the functional activity of PDI is involved in the cellular retention of TNFR:Fc protein.
Davis
R
R
Departments of Cell Science and Biochemistry, Immunex Corporation, 51 University Street, Seattle, Washington 98101, USA.
Schooley
K
K
Rasmussen
B
B
Thomas
J
J
Reddy
P
P
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D019409:Interleukin-15 / Q000235:genetics; D018613:Microscopy, Confocal; D011233:Precipitin Tests; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D007141:Immunoglobulin Fc Fragments; D019409:Interleukin-15; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases