Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris
| Autor: | Johanne Mørch Jensen, Birgit Christine Bønsager, Maher Abou Hachem, Birte Svensson, Malene Bech Vester-Christensen, Marie Sofie Møller, Hans Erik Mølager Christensen |
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| Rok vydání: | 2011 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Hot Temperature Glycoside Hydrolases Electrospray ionization Size-exclusion chromatography Cell Count Chromatography Affinity Pichia Pichia pastoris Hydrolysis chemistry.chemical_compound Bioreactors Transformation Genetic Affinity chromatography Enzyme Stability Limit dextrinase Cloning Molecular Enzyme Inhibitors Glucans Plant Proteins Chromatography biology Chemistry Pullulan Hordeum Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Kinetics Biochemistry Fermentation Seeds Chromatography Gel Biotechnology Half-Life Plasmids |
| Zdroj: | Protein expression and purification. 79(2) |
| ISSN: | 1096-0279 |
| Popis: | 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 5 L fed-batch bioreactor. Thus about 200 mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1 L 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 1:1 M 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. |
| Databáze: | OpenAIRE |
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