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Modifications of a signal sequence for antibody secretion from insect cells.

ABSTRACT: 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.

SUBMITTER: Ohmuro-Matsuyama Y

PROVIDER: S-EPMC6021281 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Modifications of a signal sequence for antibody secretion from insect cells.

Ohmuro-Matsuyama Yuki Y Yamaji Hideki H

Cytotechnology 20170605 3

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 th ...[more]

PMID: 28584932

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Project description:BackgroundSecretory signal peptides (SPs) are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1) of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv's). The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins.ResultsWe generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv's, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv's contained one additional amino-acid (AA).ConclusionsThe obtained results demonstrate the importance of SP-sequence optimization for efficient expression-secretion of scFv's. We could successfully demonstrate that minor modifications in the AA-sequence in the c-region of the natural SP from SAP1, based on in-silico predictions following the (-3, -1) rule, resulted in different expression-secretion rates of the protein of interest. The yield of scFv production could be improved close to one order of magnitude. Therefore, SP-sequence optimization is a viable option to increase the overall yield of recombinant protein production.

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Modifications of a signal sequence for antibody secretion from insect cells.

Publications

Modifications of a signal sequence for antibody secretion from insect cells.

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