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Recombinant DNA01:09

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Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Selenium incorporation using recombinant techniques.

Helen Walden1

  • 1Protein Structure and Function Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, England. helen.walden@cancer.org.uk

Acta Crystallographica. Section D, Biological Crystallography
|April 13, 2010
PubMed
Summary
This summary is machine-generated.

Selenomethionine and selenocysteine are key for phasing macromolecular structures using selenium. Recent advances in eukaryotic expression and purification techniques for seleno-labeled proteins are discussed.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Selenomethionine and selenocysteine are commonly used heavy atoms for Multi-wavelength Anomalous Dispersion (MAD) phasing in macromolecular structure determination.
  • Selenium incorporation is a well-established technique in prokaryotic expression systems.

Purpose of the Study:

  • To discuss recent advances in selenium labeling for eukaryotic expression systems.
  • To provide practical considerations for the purification and crystallization of seleno-labeled proteins.

Main Methods:

  • Recombinant techniques for selenium incorporation in prokaryotic systems.
  • Recent advancements in selenium labeling for eukaryotic expression.
  • Purification and crystallization strategies for seleno-labeled proteins.

Main Results:

  • Selenium remains the most utilized heavy atom for MAD phasing.
  • Advances in eukaryotic expression systems facilitate broader application of selenium labeling.
  • Guidance on purification and crystallization aids in successful structure determination.

Conclusions:

  • Selenomethionine and selenocysteine are crucial for macromolecular structure determination via MAD phasing.
  • Emerging techniques enhance selenium labeling in eukaryotic systems.
  • Practical advice on protein handling improves the success rate of structural studies.