Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.

<h4>Background</h4>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 fo...

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Autores principales:	Benjamin Dälken, Robert A Jabulowsky, Pranav Oberoi, Itai Benhar, Winfried S Wels
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9b058161300746e0baf7a7f5dc13a391
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spelling	oai:doaj.org-article:9b058161300746e0baf7a7f5dc13a3912021-11-18T07:01:19ZMaltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.1932-620310.1371/journal.pone.0014404https://doaj.org/article/9b058161300746e0baf7a7f5dc13a3912010-12-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21203542/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methods and findings</h4>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 α-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.<h4>Conclusions</h4>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.Benjamin DälkenRobert A JabulowskyPranav OberoiItai BenharWinfried S WelsPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 12, p e14404 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Benjamin Dälken Robert A Jabulowsky Pranav Oberoi Itai Benhar Winfried S Wels Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
description	<h4>Background</h4>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.<h4>Methods and findings</h4>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 α-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.<h4>Conclusions</h4>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.
format	article
author	Benjamin Dälken Robert A Jabulowsky Pranav Oberoi Itai Benhar Winfried S Wels
author_facet	Benjamin Dälken Robert A Jabulowsky Pranav Oberoi Itai Benhar Winfried S Wels
author_sort	Benjamin Dälken
title	Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
title_short	Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
title_full	Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
title_fullStr	Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
title_full_unstemmed	Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
title_sort	maltose-binding protein enhances secretion of recombinant human granzyme b accompanied by in vivo processing of a precursor mbp fusion protein.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/9b058161300746e0baf7a7f5dc13a391
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Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.

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