Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells.
The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains.
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Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Betenbaugh
M J
MJ
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D003720:Densitometry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006360:Heat-Shock Proteins; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics; D009036:Moths; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism*; D012995:Solubility
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007074:Immunoglobulin G; D018832:Molecular Chaperones; D011994:Recombinant Proteins