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Related Concept Videos

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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Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection
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Green fluorescence induced by EF-hand assembly in a split GFP system.

Stina Lindman1, Ida Johansson, Eva Thulin

  • 1Department of Biophysical Chemistry, Chemical Center, Lund University, Lund, Sweden. stina.lindman@bpc.lu.se

Protein Science : a Publication of the Protein Society
|May 28, 2009
PubMed
Summary

Researchers engineered a calcium-dependent green fluorescent protein (GFP) by fusing GFP fragments with calbindin. This novel system enables efficient protein reassembly and bright fluorescence, controlled by calcium levels.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Green fluorescent protein (GFP) reassembly from fragments requires high affinity, which is lacking between native GFP fragments.
  • Low affinity prevents spontaneous in vivo reassembly, chromophore maturation, and cellular fluorescence.

Purpose of the Study:

  • To engineer a calcium-dependent molecular switch for efficient GFP assembly.
  • To enhance GFP fluorescence by utilizing high-affinity protein interactions.

Main Methods:

  • Fusion of GFP fragments (1-157 and 158-238) with calbindin D(9k) EF-hands to create GFPN-EF1 and EF2-GFPC.
  • Co-expression of fusion proteins for spontaneous assembly.
  • Purification via metal-ion chelate chromatography and characterization using surface plasmon resonance (SPR).

Main Results:

  • Co-expressed GFPN-EF1 and EF2-GFPC assembled rapidly into a fluorescent protein.
  • The engineered system exhibited brighter fluorescence than control constructs.
  • Assembly and disassembly were reversible and calcium-dependent, confirmed by SPR.
  • Temperature dependence of fluorescence was calcium-dependent.

Conclusions:

  • Calbindin EF-hands facilitate efficient, calcium-dependent GFP assembly.
  • This engineered system provides a novel tool for controlled protein reassembly and fluorescence.
  • The reversible and calcium-sensitive nature offers potential for advanced biological imaging and assays.