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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

Processing of MBP-GrB fusion proteins.(A) Variants of the furin recognition motif either containing (furS) or lacking (fur) an additional C-terminal serine residue. (B) Culture supernatants of yeast clones carrying pPIC9-MBP-fur-GrB were collected after induction with methanol for 3 days, and expression and processing of the GrB fusion protein was analyzed by immunoblotting with GrB-specific antibody. (C) Samples enriched for processed (100 kDa filtrate) or unprocessed MBP-fur-GrB protein (100 kDa retentate) were prepared by filtration of culture supernatant of a representative MBP-fur-GrB clone through Amicon filters with 100 kDa membranes, and analyzed by immunoblotting with GrB-specific antibody. Initial MBP-fur-GrB containing supernatant and supernatant from an MBP-furS-GrB clone were included for comparison. Supernatant of yeast cells carrying empty pPIC9 served as a control. The lower apparent molecular weight of processed GrB in (C) when compared to (B) is most likely due to a lower degree of glycosylation (calculated molecular weight for processed GrB in non-glycosylated form: 28.4 kDa). (D) Enzymatic activity of GrB proteins from culture supernatants and kinetics of substrate cleavage were analyzed in a colorimetric peptide cleavage assay. The MBP-fur-GrB or MBP-furS-GrB containing supernatants, and the MBP-fur-GrB samples enriched for processed (100 kDa filtrate) or unprocessed protein (100 kDa retentate) analyzed in (C) were tested as indicated. A purified recombinant GrB derivative (GrB) was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 displayed no GrB activity (data not shown). Means of triplicate samples are shown. Error bars indicate SEM.
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pone-0014404-g005: Processing of MBP-GrB fusion proteins.(A) Variants of the furin recognition motif either containing (furS) or lacking (fur) an additional C-terminal serine residue. (B) Culture supernatants of yeast clones carrying pPIC9-MBP-fur-GrB were collected after induction with methanol for 3 days, and expression and processing of the GrB fusion protein was analyzed by immunoblotting with GrB-specific antibody. (C) Samples enriched for processed (100 kDa filtrate) or unprocessed MBP-fur-GrB protein (100 kDa retentate) were prepared by filtration of culture supernatant of a representative MBP-fur-GrB clone through Amicon filters with 100 kDa membranes, and analyzed by immunoblotting with GrB-specific antibody. Initial MBP-fur-GrB containing supernatant and supernatant from an MBP-furS-GrB clone were included for comparison. Supernatant of yeast cells carrying empty pPIC9 served as a control. The lower apparent molecular weight of processed GrB in (C) when compared to (B) is most likely due to a lower degree of glycosylation (calculated molecular weight for processed GrB in non-glycosylated form: 28.4 kDa). (D) Enzymatic activity of GrB proteins from culture supernatants and kinetics of substrate cleavage were analyzed in a colorimetric peptide cleavage assay. The MBP-fur-GrB or MBP-furS-GrB containing supernatants, and the MBP-fur-GrB samples enriched for processed (100 kDa filtrate) or unprocessed protein (100 kDa retentate) analyzed in (C) were tested as indicated. A purified recombinant GrB derivative (GrB) was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 displayed no GrB activity (data not shown). Means of triplicate samples are shown. Error bars indicate SEM.

Mentions: GrB is naturally expressed as a pre-pro-enzyme, that requires stepwise removal of the signal peptide and an N-terminal activation dipeptide to yield active protein [31]. Hence, for expression of recombinant GrB in P. pastoris we previously fused the sequence of mature GrB directly to the α-factor signal peptide in pPIC9-GrB, allowing secretion of fully processed and active enzyme into the culture supernatant [24]. In contrast to the minimal furin recognition motif Arg-X-X-Arg which is cleaved after the C-terminal arginine residue, the optimized furS cleavage site contains an additional C-terminal serine residue which is present in many physiological substrates of furin [26], and resides at the N-terminus of the cleavage product after processing (Fig. 5A). To enable production of GrB with an N-terminus identical to that of the natural protein, we generated a modified pPIC9-MBP-fur-GrB construct which carries a furin recognition motif (fur) lacking the C-terminal serine of furS.


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)

Processing of MBP-GrB fusion proteins.(A) Variants of the furin recognition motif either containing (furS) or lacking (fur) an additional C-terminal serine residue. (B) Culture supernatants of yeast clones carrying pPIC9-MBP-fur-GrB were collected after induction with methanol for 3 days, and expression and processing of the GrB fusion protein was analyzed by immunoblotting with GrB-specific antibody. (C) Samples enriched for processed (100 kDa filtrate) or unprocessed MBP-fur-GrB protein (100 kDa retentate) were prepared by filtration of culture supernatant of a representative MBP-fur-GrB clone through Amicon filters with 100 kDa membranes, and analyzed by immunoblotting with GrB-specific antibody. Initial MBP-fur-GrB containing supernatant and supernatant from an MBP-furS-GrB clone were included for comparison. Supernatant of yeast cells carrying empty pPIC9 served as a control. The lower apparent molecular weight of processed GrB in (C) when compared to (B) is most likely due to a lower degree of glycosylation (calculated molecular weight for processed GrB in non-glycosylated form: 28.4 kDa). (D) Enzymatic activity of GrB proteins from culture supernatants and kinetics of substrate cleavage were analyzed in a colorimetric peptide cleavage assay. The MBP-fur-GrB or MBP-furS-GrB containing supernatants, and the MBP-fur-GrB samples enriched for processed (100 kDa filtrate) or unprocessed protein (100 kDa retentate) analyzed in (C) were tested as indicated. A purified recombinant GrB derivative (GrB) was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 displayed no GrB activity (data not shown). Means of triplicate samples are shown. Error bars indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008669&req=5

pone-0014404-g005: Processing of MBP-GrB fusion proteins.(A) Variants of the furin recognition motif either containing (furS) or lacking (fur) an additional C-terminal serine residue. (B) Culture supernatants of yeast clones carrying pPIC9-MBP-fur-GrB were collected after induction with methanol for 3 days, and expression and processing of the GrB fusion protein was analyzed by immunoblotting with GrB-specific antibody. (C) Samples enriched for processed (100 kDa filtrate) or unprocessed MBP-fur-GrB protein (100 kDa retentate) were prepared by filtration of culture supernatant of a representative MBP-fur-GrB clone through Amicon filters with 100 kDa membranes, and analyzed by immunoblotting with GrB-specific antibody. Initial MBP-fur-GrB containing supernatant and supernatant from an MBP-furS-GrB clone were included for comparison. Supernatant of yeast cells carrying empty pPIC9 served as a control. The lower apparent molecular weight of processed GrB in (C) when compared to (B) is most likely due to a lower degree of glycosylation (calculated molecular weight for processed GrB in non-glycosylated form: 28.4 kDa). (D) Enzymatic activity of GrB proteins from culture supernatants and kinetics of substrate cleavage were analyzed in a colorimetric peptide cleavage assay. The MBP-fur-GrB or MBP-furS-GrB containing supernatants, and the MBP-fur-GrB samples enriched for processed (100 kDa filtrate) or unprocessed protein (100 kDa retentate) analyzed in (C) were tested as indicated. A purified recombinant GrB derivative (GrB) was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 displayed no GrB activity (data not shown). Means of triplicate samples are shown. Error bars indicate SEM.
Mentions: GrB is naturally expressed as a pre-pro-enzyme, that requires stepwise removal of the signal peptide and an N-terminal activation dipeptide to yield active protein [31]. Hence, for expression of recombinant GrB in P. pastoris we previously fused the sequence of mature GrB directly to the α-factor signal peptide in pPIC9-GrB, allowing secretion of fully processed and active enzyme into the culture supernatant [24]. In contrast to the minimal furin recognition motif Arg-X-X-Arg which is cleaved after the C-terminal arginine residue, the optimized furS cleavage site contains an additional C-terminal serine residue which is present in many physiological substrates of furin [26], and resides at the N-terminus of the cleavage product after processing (Fig. 5A). To enable production of GrB with an N-terminus identical to that of the natural protein, we generated a modified pPIC9-MBP-fur-GrB construct which carries a furin recognition motif (fur) lacking the C-terminal serine of furS.

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