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Monoclonal antibodies and antibody fragments have recently been developed for use in diverse diagnostic and therapeutic applications. Insect cells can efficiently secrete recombinant proteins such as antibody molecules through post-translational processing and modifications that are similar to those performed in mammalian cells. In eukaryotic cells, the signal sequence in a nascent polypeptide is recognized by the signal recognition particle, and the polypeptide is then folded and modified in the endoplasmic reticulum. The signal sequence consists of three regions, a positively charged N-terminus, a hydrophobic core, and a polar C-terminus. In the present study, we examined the substitutions of the characteristic amino acids of a Drosophila immunoglobulin heavy chain binding protein signal sequence, and investigated the effect on the secretory production of an antibody Fab fragment from lepidopteran insect cells in transient expression. A modification of the signal sequence for the heavy chain resulted in a twofold increase in the secreted Fab fragment, while the modification for the light chain led to a more than 3.6-fold increase. [ABSTRACT FROM AUTHOR]

Abstract: Protein–protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein–protein interaction assay, the firefly luminescent intermediate‐based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half‐reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin‐induced FKBP12–FRB interactions. [ABSTRACT FROM AUTHOR]

Protein-protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein-protein interaction assay, the firefly luminescent intermediate-based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity ('Acceptor' Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12-FRB interactions., (Copyright © 2017 John Wiley & Sons, Ltd.)