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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.

Dälken B, Jabulowsky RA, Oberoi P, Benhar I, Wels WS - PLoS ONE (2010)

Bottom Line: MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST.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.

View Article: PubMed Central - PubMed

Affiliation: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, Germany.

ABSTRACT

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 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.

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.

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Related in: MedlinePlus

Secretion of GST and MBP into the culture medium.The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A.
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pone-0014404-g004: Secretion of GST and MBP into the culture medium.The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A.

Mentions: To investigate the fate of GST and MBP domains after expression of the fusion proteins, we examined culture supernatants by immunoblot analysis with GST- and MBP-specific antibodies. Thereby no unprocessed GST-furS-GrB (Fig. 4A) or MBP-furS-GrB (Fig. 4B) proteins were detected, confirming the results with GrB-specific antibody shown above. However, we observed an increase over time of soluble GST and MBP, that reflected the previously observed levels of processed GrB (see Fig. 2A for comparison). The apparent molecular weights of the single bands detected correspond well with the calculated molecular weights of the MBP (40.6 kDa) and GST (25.6 kDa) moieties. This indicates complete processing of the fusion proteins by cleavage within the furS recognition site in the secretory pathway, resulting in secretion of processed GrB and both fusion partners GST and MBP.


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

Dälken B, Jabulowsky RA, Oberoi P, Benhar I, Wels WS - PLoS ONE (2010)

Secretion of GST and MBP into the culture medium.The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008669&req=5

pone-0014404-g004: Secretion of GST and MBP into the culture medium.The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A.
Mentions: To investigate the fate of GST and MBP domains after expression of the fusion proteins, we examined culture supernatants by immunoblot analysis with GST- and MBP-specific antibodies. Thereby no unprocessed GST-furS-GrB (Fig. 4A) or MBP-furS-GrB (Fig. 4B) proteins were detected, confirming the results with GrB-specific antibody shown above. However, we observed an increase over time of soluble GST and MBP, that reflected the previously observed levels of processed GrB (see Fig. 2A for comparison). The apparent molecular weights of the single bands detected correspond well with the calculated molecular weights of the MBP (40.6 kDa) and GST (25.6 kDa) moieties. This indicates complete processing of the fusion proteins by cleavage within the furS recognition site in the secretory pathway, resulting in secretion of processed GrB and both fusion partners GST and MBP.

Bottom Line: MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST.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.

View Article: PubMed Central - PubMed

Affiliation: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, Germany.

ABSTRACT

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 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.

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.

Show MeSH
Related in: MedlinePlus