title: Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp.
abstract: A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.
authors.affiliation: Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Aoki
authors.forename: Md
authors.initials: M
authors.lastname: Nazmul Ahsan
authors.forename: Shugo
authors.initials: S
authors.lastname: Watabe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D033542:Pandalidae / Q000201:enzymology*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases