A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells.
The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase from Bacillus subtilis (SipS) was constructed for expression studies. When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates. Conversely, coexpression of scFv alone or with an inactive mutant SipS resulted in at least 30% of the intracellular polypeptide in an unprocessed form at 3 days post infection. Production of scFv in the medium was also enhanced in the presence of SipS; however, low secretion levels indicate the presence of a post-processing bottleneck.
Ailor
E
E
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2494, USA.
Pathmanathan
J
J
Jongbloed
J D
JD
Betenbaugh
M J
MJ
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D005822:Genetic Vectors / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase