Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

Johanne Mørch Jensen, Malene Bech Vester-Christensen, Marie Sofie Møller, Birgit Christine Bønsager, Hans Erik Mølager Christensen, Maher Abou Hachem, Birte Svensson

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.
Original languageEnglish
JournalProtein Expression and Purification
Volume79
Issue number2
Pages (from-to)217-222
ISSN1046-5928
DOIs
Publication statusPublished - 2011

Keywords

  • Pullulan
  • High stability
  • Fed-batch bioreactor fermentation
  • Limit dextrinase inhibitor
  • Electrospray ionization mass spectrometry

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