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Related Experiment Videos

Shotgun phage display cloning.

K Jacobsson1, L Frykberg

  • 1Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, UPPSALA, SE-750 07 UPPSALA, Sweden. Karin.Jacobsson@mikrob.slu.se

Combinatorial Chemistry & High Throughput Screening
|April 3, 2001
PubMed
Summary

Shotgun phage display cloning effectively identifies bacterial-host protein interactions. Gene VIII-based display significantly enhances the frequency of correct clones for Staphylococcus aureus proteins.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Shotgun phage display cloning is a valuable technique for investigating protein interactions.
  • Libraries are created by fragmenting prokaryotic DNA and cloning it into phage vectors.
  • Theoretically, these libraries can represent all proteins encoded by a bacterial genome.

Purpose of the Study:

  • To evaluate the efficacy of shotgun phage display cloning for identifying Staphylococcus aureus protein interactions.
  • To compare the performance of gene III-based and gene VIII-based phage display systems.

Main Methods:

  • Construction of gene III-based and gene VIII-based phage display libraries from Staphylococcus aureus DNA.
  • Selection of libraries against immobilized IgG and fibronectin.
  • Analysis of positive clones after multiple panning rounds.

Main Results:

  • A gene III-based library yielded 20-40% positive clones after two panning rounds.
  • Switching to a gene VIII-based display, which increases fusion proteins per phage, raised positive clone frequency to 75-100%.

Conclusions:

  • Shotgun phage display is a powerful tool for studying bacterial-host protein interactions.
  • Gene VIII-based display significantly improves the efficiency and success rate of identifying target proteins compared to gene III-based display.

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