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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{"title"=>"Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein", "type"=>"journal", "authors"=>[{"first_name"=>"Benjamin", "last_name"=>"Dälken", "scopus_author_id"=>"12784584400"}, {"first_name"=>"Robert A.", "last_name"=>"Jabulowsky", "scopus_author_id"=>"36731091600"}, {"first_name"=>"Pranav", "last_name"=>"Oberoi", "scopus_author_id"=>"34968522600"}, {"first_name"=>"Itai", "last_name"=>"Benhar", "scopus_author_id"=>"6701578664"}, {"first_name"=>"Winfried S.", "last_name"=>"Wels", "scopus_author_id"=>"7004562782"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-78650989328", "sgr"=>"78650989328", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0014404", "pmid"=>"21203542", "pui"=>"361059732"}, "id"=>"d8579ed6-8e85-3fe0-9459-24f5d82fe7cf", "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.\\n\\nMETHODS 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.\\n\\nCONCLUSIONS: 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.", "link"=>"http://www.mendeley.com/research/maltosebinding-protein-enhances-secretion-recombinant-human-granzyme-b-accompanied-vivo-processing-p", "reader_count"=>25, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>5, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>2, "Student > Master"=>2, "Student > Bachelor"=>3, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>5, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>2, "Student > Master"=>2, "Student > Bachelor"=>3, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>4, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>15, "Medicine and Dentistry"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>4}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Austria"=>2}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/810529"], "description"=>"<p>(A) Cultures of <i>P. pastoris</i> carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector were induced with methanol. Samples were taken every 24 h, and OD<sub>600</sub> was determined as a measure for cell density. (B) Quantification of GrB mRNA levels. RNA was isolated from yeast cells carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector after induction with methanol for one day. cDNA was synthesized and analyzed by quantitative real-time PCR including GAPDH for normalization. Means of duplicate samples are shown. Error bars indicate SD.</p>", "links"=>[], "tags"=>["kinetics", "yeast", "clones", "grb", "mrna"], "article_id"=>480897, "categories"=>["Biotechnology"], "users"=>["Benjamin Dälken", "Robert A. Jabulowsky", "Pranav Oberoi", "Itai Benhar", "Winfried S. Wels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014404.g003", "stats"=>{"downloads"=>2, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Growth_kinetics_of_yeast_clones_and_GrB_mRNA_expression_/480897", "title"=>"Growth kinetics of yeast clones and GrB mRNA expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:39:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/810273"], "description"=>"<p>(A) Constructs for expression of human granzyme B, and MBP-GrB and GST-GrB fusion proteins. <i>AOX1</i>, methanol-inducible alcohol oxidase I promoter; SP, α-factor signal peptide; furS, furin recognition motif; M, Myc tag; H, polyhistidine tag. (B) Culture supernatants of yeast clones carrying pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected after induction with methanol for 3 days, and expression of GrB was analyzed by immunoblotting with GrB-specific antibody. Each lane represents an individual clone. Clones marked with an asterisk were used in subsequent experiments. (C) For comparison, culture supernatants of clones carrying the different expression constructs were analyzed together in a single immunoblot experiment as indicated. Supernatant of a yeast clone carrying empty pPIC9 served as a control.</p>", "links"=>[], "tags"=>["grb", "yeast"], "article_id"=>480635, "categories"=>["Biotechnology"], "users"=>["Benjamin Dälken", "Robert A. Jabulowsky", "Pranav Oberoi", "Itai Benhar", "Winfried S. Wels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014404.g001", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_GrB_in_the_yeast_Pichia_pastoris_/480635", "title"=>"Expression of GrB in the yeast <i>Pichia pastoris</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:38:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/810743"], "description"=>"<p>(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.</p>", "links"=>[], "tags"=>["mbp-grb", "fusion"], "article_id"=>481110, "categories"=>["Biotechnology"], "users"=>["Benjamin Dälken", "Robert A. Jabulowsky", "Pranav Oberoi", "Itai Benhar", "Winfried S. Wels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014404.g005", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Processing_of_MBP_GrB_fusion_proteins_/481110", "title"=>"Processing of MBP-GrB fusion proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:40:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/810412"], "description"=>"<p>(A) Supernatants of yeast cultures harboring pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected at the indicated time points after induction with methanol, and analyzed by immunoblotting with GrB-specific antibody. Band intensities were quantified by determining mean gray values (MGV) relative to the highest value obtained. (B) Expression of an ErbB2 protein fragment. Yeast cells carrying pPIC9-ErbB2<sub>222</sub> or pPIC9-MBP-furS-ErbB2<sub>222</sub> were induced with methanol, and ErbB2 protein in culture supernatants was analyzed by immunoblotting with ErbB2-specific antibody.</p>", "links"=>[], "tags"=>["grb", "secreted"], "article_id"=>480775, "categories"=>["Biotechnology"], "users"=>["Benjamin Dälken", "Robert A. Jabulowsky", "Pranav Oberoi", "Itai Benhar", "Winfried S. Wels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014404.g002", "stats"=>{"downloads"=>3, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_GrB_secreted_into_the_culture_medium_/480775", "title"=>"Analysis of GrB secreted into the culture medium.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:38:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/810919"], "description"=>"<p>(A) Culture supernatants of representative yeast clones carrying pPIC9-MBP-fur-GST or pPIC9-MBP-furS-GST were collected after induction with methanol for 3 days, and expression and processing of the MBP-GST fusion proteins was analyzed by immunoblotting with GST-specific (left panel) or MBP-specific antibody (right panel). Supernatant of yeast cells carrying empty pPIC9 served as a control. (B) Enzymatic activity of MBP-GST fusion proteins and kinetics of conjugation of glutathione to synthetic CDNB substrate was analyzed in a colorimetric assay. The MBP-fur-GST or MBP-furS-GST containing supernatants analyzed in (A) were tested as indicated. Commercially available recombinant GST was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 served as negative control. Means of triplicate samples are shown. Error bars indicate SEM.</p>", "links"=>[], "tags"=>["enzymatic", "mbp-gst", "fusion"], "article_id"=>481287, "categories"=>["Biotechnology"], "users"=>["Benjamin Dälken", "Robert A. Jabulowsky", "Pranav Oberoi", "Itai Benhar", "Winfried S. Wels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014404.g006", "stats"=>{"downloads"=>2, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Processing_and_enzymatic_activity_of_MBP_GST_fusion_proteins_/481287", "title"=>"Processing and enzymatic activity of MBP-GST fusion proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:41:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/810613"], "description"=>"<p>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 <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014404#pone-0014404-g002\" target=\"_blank\">Fig. 2A</a>.</p>", "links"=>[], "tags"=>["gst", "mbp"], "article_id"=>480983, "categories"=>["Biotechnology"], "users"=>["Benjamin Dälken", "Robert A. Jabulowsky", "Pranav Oberoi", "Itai Benhar", "Winfried S. Wels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014404.g004", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Secretion_of_GST_and_MBP_into_the_culture_medium_/480983", "title"=>"Secretion of GST and MBP into the culture medium.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:40:04"}

PMC Usage Stats | Further Information

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