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Related Concept Videos

Recombinant DNA01:09

Recombinant DNA

Overview
Reporter Genes02:11

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...

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Expression and characterization of recombinant ecarin.

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Recombinant ecarin, a snake venom protease, was successfully produced in CHO-S cells. This recombinant form shows enhanced prothrombin conversion, making it a promising replacement for native ecarin in assays.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Ecarin is a snake venom protease crucial for prothrombin activation.
  • Native ecarin assays are vital for hemostasis diagnostics.
  • Production challenges exist for native ecarin.

Purpose of the Study:

  • To develop a recombinant ecarin (r-ecarin) using CHO-S cells.
  • To characterize the enzymatic activity of r-ecarin.
  • To evaluate r-ecarin as a substitute for native ecarin in assays.

Main Methods:

  • Stable transfection of CHO-S cells for ecarin expression.
  • Culturing cells in animal component-free medium.
  • Assessing r-ecarin activity using prothrombin and prethrombin-2 substrates and chromogenic assays.

Main Results:

  • Recombinant ecarin was secreted as a pro-protein and auto-activated post-cell death.
  • Maximal activity was achieved 7 days after cell viability dropped to zero.
  • r-ecarin demonstrated a two-fold higher turnover rate for prothrombin compared to native ecarin at concentrations above 250 nM.
  • Kinetic parameters (Km) were determined for r-ecarin with prethrombin-2 (0.4 μM) and prothrombin (approx. 0.9 μM).

Conclusions:

  • Recombinant ecarin can be effectively produced in CHO-S cells.
  • r-ecarin exhibits superior catalytic efficiency for prothrombin conversion.
  • r-ecarin is a viable and promising alternative to native ecarin for diagnostic and research applications.