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Related Experiment Videos

Purification and biochemical characterization of the lambda holin

D L Smith1, D K Struck, J M Scholtz

  • 1Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.

Journal of Bacteriology
|May 9, 1998
PubMed
Summary

Lambda phage holins, like S105, are proteins that create temporary holes in membranes. Researchers purified S105 using a histidine tag, confirming its ability to form membrane pores essential for phage lysis.

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Area of Science:

  • Molecular biology
  • Biochemistry
  • Membrane protein research

Background:

  • Holins are essential phage proteins that induce host cell lysis by permeabilizing the cytoplasmic membrane.
  • The lambda phage gene S encodes holin proteins, with S105 being a key example studied for its lytic function.

Purpose of the Study:

  • To purify and characterize the lambda phage S105 holin protein.
  • To confirm the role of S105 in forming membrane pores responsible for phage-induced lysis.

Main Methods:

  • Oligohistidine tagging of the S105 protein for enhanced purification via immobilized metal affinity chromatography.
  • Overproduction and purification of the tagged S105 protein.
  • Circular dichroism spectroscopy to determine protein secondary structure.

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  • In vitro liposome-based assay to assess membrane permeabilization activity.

    • Main Results:

    • A functional S105 holin allele (S105H94) was successfully purified using affinity chromatography.
    • Circular dichroism revealed approximately 40% alpha-helix content, supporting a transmembrane helical structure.
    • Purified S105 induced the release of encapsulated dye from liposomes, demonstrating pore-forming activity.
    • A mutant S protein lacking pore-forming ability did not induce dye release in the liposome assay.

    Conclusions:

    • The lambda phage S protein functions as a bitopic membrane protein.
    • S105 is capable of forming aqueous holes in lipid bilayers, a critical step in the phage lysis cycle.