Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P11021 (BiP)
 2,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t-PA) was expressed, N-glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214-223, 1989). We also showed that t-PA secretion was blocked by tunicamycin (TM), an inhibitor of N-glycosylation, but not by castanospermine (CS) or N-methyldeoxynojirimycin, inhibitors of the initial steps in N-linked oligosaccharide processing. This suggested that the addition, but not the processing, of N-linked oligosaccharides is required for the secretion of recombinant t-PA from baculovirus-infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N-glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane-bound glycoproteins were expressed in control, TM-treated, or CS-treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N-glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM-treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM-treated Sf9 cells by binding to them and producing transport-incompetent heterooligomeric complexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose-regulated 78 kilodalton protein (BiP/GRP78).
 ...
PMID:Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells. 234 87

Foreign secretory pathway proteins are often produced in surprisingly low amounts in the baculovirus/insect cell expression system. One possible reason for this is that heterologous signal peptides might be inefficiently recognized by the insect cell protein translocation machinery. This idea was supported by a recent study showing that secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide (Tessier, D. C., Thomas, D. Y., Khouri, H. E., Laliberte, F., and Vernet, T. (1991) Gene (Amst.) 98, 177-183). We have extended these observations by measuring the effects of different signal peptide and signal peptide-prosequence combinations on baculovirus-mediated expression and secretion of human tissue plasminogen activator (t-PA). Replacement of the native prepropeptide with signal peptides from a lepidopteran insect secretory protein (cecropin B), a major baculovirus structural glycoprotein (64K), or an abundant, highly conserved lumenal protein of the rough endoplasmic reticulum (GRP78/BiP, a 78-kDa glucose-regulated protein/immunoglobulin heavy chain-binding protein), had no significant effect on t-PA expression or secretion. The same results were obtained with the signal peptide from honeybee prepromellitin, which was able to enhance secretion of plant propapain (Tessier et al., 1991 (above)). Similar results were obtained when heterologous signal peptides were combined with the native prosequence or when the intact cecropin B preprosequence was used. Translational initiation at an upstream, in-frame ATT, which could functionally inactivate any signal peptide, did not explain the low efficiency of t-PA secretion. Finally, deletion of the native signal peptide, prosequence, or both, failed to increase t-PA production. These results showed that insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. They also suggested that the inability of insect cells to recognize the processing signals in human t-PA efficiently is probably not the major factor preventing its high level production in this system.
 ...
PMID:Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. 834 55