Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein
- Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal alpha-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.
Author: | Benjamin Dälken, Robert Alexander JabulowskyGND, Pranav OberoiGND, Itai Benhar, Winfried WelsORCiDGND |
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URN: | urn:nbn:de:hebis:30-114362 |
DOI: | https://doi.org/10.1371/journal.pone.0014404 |
ISSN: | 1932-6203 |
Parent Title (English): | PLoS One |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2010/12/22 |
Date of first Publication: | 2010/12/22 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2011/09/05 |
Volume: | 5 |
Issue: | (12): e14404 |
Note: | Copyright: © 2010 Dälken et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
Source: | PLoS ONE 5(12): e14404. doi: 10.1371/journal.pone.0014404 |
HeBIS-PPN: | 276028074 |
Institutes: | Medizin |
Angeschlossene und kooperierende Institutionen / Georg-Speyer-Haus | |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Sammlung Biologie / Sondersammelgebiets-Volltexte | |
Licence (German): | Creative Commons - Namensnennung 3.0 |