title: Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli.
abstract: Escherichia coli is the best-established microbial host strain for production of proteins and chemicals, but has a weakness for not secreting high amounts of active heterologous proteins to the extracellular culture medium, of which origins belong to whether prokaryotes or eukaryotes. In this study, Candida antarctica lipase B (CalB), a popular eukaryotic enzyme which catalyzes a number of biochemical reactions and barely secreted extracellularly, was expressed functionally at a gram scale in culture medium by using a simple amino acid-tag system of E. coli. New fusion tag systems consisting of a pelB signal sequence and various anion amino acid tags facilitated both intracellular expression and extracellular secretion of CalB. Among them, the N-terminal five aspartate tag changed the quaternary structure of the dimeric CalB and allowed production of 1.9 g/L active CalB with 65 U/mL activity in culture medium, which exhibited the same enzymatic properties as the commercial CalB. This PelB-anion amino acid tag-based expression system for CalB can be extended to production of other industrial proteins hardly expressed and exported from E. coli, thereby increasing target protein concentrations and minimizing purification steps.
authors.affiliation: Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul, 151-921, Republic of Korea.
authors.forename: Sun-Ki
authors.initials: SK
authors.lastname: Kim
authors.forename: Yong-Cheol
authors.initials: YC
authors.lastname: Park
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
authors.forename: Seung Taeg
authors.initials: ST
authors.lastname: Jeon
authors.forename: Won-Ki
authors.initials: WK
authors.lastname: Min
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica
keywords: Candida antarctica lipase B; Escherichia coli; extracellular secretion; polyanionic amino acid tag; quaternary structure change