Rescue of immunoglobulins from insolubility is facilitated by PDI in the baculovirus expression system.
A substantial fraction of immunoglobulin heavy and light chain polypeptides were insoluble when expressed in the baculovirus-insect cell expression system. In the presence of coexpressed heterologous protein disulfide isomerase (PDI), however, the solubility of the immunoglobulins was enhanced and IgG was secreted at higher levels from baculovirus-infected Trichoplusia ni insect cells. Pulse-chase experiments indicated that some immunoglobulin polypeptides were initially insoluble in the presence of PDI but subsequently were rescued in a soluble form competent for IgG assembly and secretion. Recovery of the insoluble immunoglobulins was not observed in the absence of coexpressed PDI. Even after treatment of insect cells with tunicamycin to inhibit N-glycosylation of immunoglobulin heavy chains, coexpressed PDI was able to salvage insoluble immunoglobulins and secrete these modified glycoforms. The capacity for PDI to rescue immunoglobulins was also demonstrated in vitro where immunoglobulin heavy chains and light chain dimers were salvaged from aggregates of denatured IgG. PDI-mediated rescue of proteins, perhaps assisted by chaperones and other foldases, may be important in vivo where insolubility is a common occurrence for newly synthesized polypeptides.
Hsu
T A
TA
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2694, USA.
Watson
S
S
Eiden
J J
JJ
Betenbaugh
M J
MJ
D000818:Animals; D002478:Cells, Cultured; D005822:Genetic Vectors; D006041:Goats; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000235:genetics; D007313:Insecta / Q000378:metabolism / Q000821:virology; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014415:Tunicamycin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases