Related Experiment Video

Updated: Aug 9, 2026

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells
09:14

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Published on: August 20, 2018

Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus

T Kupke1, M Lechner, G Kaim

  • 1Institut für Mikrobielle Genetik, Universität Tübingen, Federal Republic of Germany.

European Journal of Biochemistry
|October 20, 1989
PubMed

Insights

Monophosphatidylinositol inositol phosphohydrolase (PtdIns-PLC) was purified from Bacillus thuringiensis and recombinant Escherichia coli. This enzyme, crucial for cell membrane function, showed identical amino-terminal sequences and was inhibited by divalent cations.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Monophosphatidylinositol inositol phosphohydrolase (PtdIns-PLC) plays a role in cell membrane signaling and turnover.
  • Understanding PtdIns-PLC function requires its purification and characterization.

Purpose of the Study:

  • To purify and characterize PtdIns-PLC from Bacillus thuringiensis and a recombinant Escherichia coli clone.
  • To compare the properties of PtdIns-PLC from both sources.

Main Methods:

  • Two-step purification involving ion-exchange chromatography (DEAE-Sepharose) and FPLC (Mono-Q).
  • Molecular mass determination by SDS-PAGE.
  • Isoelectric point determination.
  • Amino-terminal sequencing.
  • Enzyme activity assay using mixed micelles and activity staining.

Main Results:

  • PtdIns-PLC was successfully purified from both B. thuringiensis culture supernatant and recombinant E. coli cellular fraction.
  • Purification yields were 32% and 50% for B. thuringiensis and E. coli, respectively.
  • The enzyme exhibited a molecular mass of 34 kDa and an isoelectric point of 5.15.
  • Identical amino-terminal sequences were observed for both purified enzymes.
  • PtdIns-PLC activity was inhibited by divalent cations.
  • Enzyme activity was confirmed via gel activity staining.

Conclusions:

  • The gene for B. thuringiensis PtdIns-PLC can be expressed in E. coli, yielding a functionally similar enzyme.
  • The purified PtdIns-PLC is a 34 kDa protein with an isoelectric point of 5.15.
  • Divalent cations are inhibitors of PtdIns-PLC activity.
  • The study provides a basis for further investigation into the structure-function relationship of PtdIns-PLC.

More Related Videos

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry08:07

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA210:31

A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA2

Published on: September 26, 2025

Related Concept Videos

Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
Production of Pharmaceuticals01:30

Production of Pharmaceuticals

Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...
Production of Biopesticides01:18

Production of Biopesticides

Biopesticides offer a sustainable alternative to chemical pesticides, utilizing microbial agents to control agricultural pests. Bacillus thuringiensis (Bt) is a widely employed bacterium known for its potent insecticidal activity. Bt biopesticides are favored for their specificity to insect pests, minimal environmental impact, and natural degradability.Mechanism of Bt Toxin Action Bt produces insecticidal crystal (Cry) proteins during its sporulation phase. These proteins form parasporal...