title: Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.
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.
  
  
  RESULTS
  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.
authors.affiliation: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dälken
authors.forename: Robert A
authors.initials: RA
authors.lastname: Jabulowsky
authors.forename: Pranav
authors.initials: P
authors.lastname: Oberoi
authors.forename: Itai
authors.initials: I
authors.lastname: Benhar
authors.forename: Winfried S
authors.initials: WS
authors.lastname: Wels
mesh_terms: D020816:Amino Acid Motifs; D017209:Apoptosis; D003062:Codon; D045683:Furin / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000378:metabolism; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology; D058619:Maltose-Binding Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D003062:Codon; D058619:Maltose-Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D053804:Granzymes; D045683:Furin