Journal article
Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris
Department of Systems Biology, Technical University of Denmark1
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark2
Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark3
Department of Chemistry, Technical University of Denmark4
The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14kDa 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-His6.
At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5–10nM LD. LDI retained stability in the pH 2–12 range and at pH 6.5 displayed a half-life of 53 and 33min 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.
Language: | English |
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Year: | 2011 |
Pages: | 217-222 |
ISSN: | 10960279 and 10465928 |
Types: | Journal article |
DOI: | 10.1016/j.pep.2011.04.009 |
ORCIDs: | Møller, Marie Sofie , Christensen, Hans Erik Mølager , Abou Hachem, Maher and Svensson, Birte |
Electrospray ionization mass spectrometry Fed-batch bioreactor fermentation High stability Limit dextrinase inhibitor Pullulan
Bioreactors Cell Count Chromatography, Affinity Chromatography, Gel Cloning, Molecular Enzyme Inhibitors Enzyme Stability Fermentation Glucans Glycoside Hydrolases Half-Life Hordeum Hot Temperature Hydrogen-Ion Concentration Hydrolysis Kinetics Pichia Plant Proteins Plasmids Recombinant Proteins Seeds Spectrometry, Mass, Electrospray Ionization Transformation, Genetic pullulan pullulanase