Cellular processing limits the heterologous expression of secretory component in mammalian cells.
Recombinant vaccinia-virus-based expression systems are very popular for the overproduction of proteins in mammalian cell lines. Both the double virus T7/vaccinia hybrid system and the single recombinant strategy based on the p11 K late promoter were evaluated for their ability to govern expression and secretion of recombinant human secretory component (SC), a glycoprotein associated with IgA in mucosal secretions. We report here that, while the T7 promoter is transcriptionally 3.4-fold more active than the p11 K promoter, no difference in levels of secreted recombinant human SC is observed using either vaccinia system to infect CV-1 cells. High transcription, and thus translation levels, lead to saturation of early processing steps involved in protein export. Both systems exhibit transient accumulation of comparable amount of recombinant human SC in the endoplasmic reticulum and/or the cis Golgi network, as demonstrated by immunofluorescence and endoglycosidase H (EndoH) sensitivities. Exposure of infected cells to tunicamycin results in similar inhibition of recombinant human SC export, further arguing that N-linked glycosylation is necessary for proper folding and subsequent secretion. Moreover, pulse-chase experiments indicate that newly synthesized recombinant human SC is not completely processed in a mature glycoprotein and that a portion of overexpressed SC might be degraded before it can be secreted. Recombinant human SC behaves identically to native SC in terms of kinetics of secretion and IgA-binding capacity. Our results indicate that optimization of expression systems should not only rely on the design of effective vectors, but also on the identification and clearance of the cellular bottlenecks associated with maturation of the secreted proteins.
Cottet
S
S
ISREC, Epalinges, Switzerland.
Corthésy
B
B
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D017123:Bacteriophage T7 / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D015870:Gene Expression; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012635:Secretory Component / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology; D014616:Vaccinia virus / Q000235:genetics
D000998:Antiviral Agents; D011994:Recombinant Proteins; D012635:Secretory Component; D014415:Tunicamycin