title: Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris.
abstract: The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Johanne Mørch
authors.initials: JM
authors.lastname: Jensen
authors.forename: Malene Bech
authors.initials: MB
authors.lastname: Vester-Christensen
authors.forename: Marie Sofie
authors.initials: MS
authors.lastname: Møller
authors.forename: Birgit C
authors.initials: BC
authors.lastname: Bønsager
authors.forename: Hans Erik Mølager
authors.initials: HE
authors.lastname: Christensen
authors.forename: Maher Abou
authors.initials: MA
authors.lastname: Hachem
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D019149:Bioreactors; D002452:Cell Count; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004795:Enzyme Stability; D005285:Fermentation; D005936:Glucans / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006207:Half-Life; D001467:Hordeum / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D010843:Pichia; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D012639:Seeds / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D014170:Transformation, Genetic
chemical_list: D004791:Enzyme Inhibitors; D005936:Glucans; D010940:Plant Proteins; D011994:Recombinant Proteins; C009109:pullulan; D006026:Glycoside Hydrolases; C100181:pullulanase