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Related Experiment Videos

Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display.

John Löfblom1, Henrik Wernérus, Stefan Ståhl

  • 1Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.

FEMS Microbiology Letters
|June 21, 2005
PubMed
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This study presents a novel staphylococcal surface display system for protein library selection. A normalization strategy using a generic protein tag enables fine affinity discrimination, crucial for discovering improved protein variants.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Combinatorial biochemistry requires protein library display systems for efficient selection.
  • Fine affinity discrimination is essential to avoid losing improved protein variants during selection.
  • Cell-to-cell variations in surface expression can bias selection outcomes.

Purpose of the Study:

  • To investigate a staphylococcal surface display system for protein library display.
  • To develop a method for normalizing target-binding signals to avoid expression level biases.
  • To evaluate the system's capability for fine affinity discrimination of protein variants.

Main Methods:

  • Engineered Staphylococcus carnosus cells displaying mutated staphylococcal protein A variants.

Related Experiment Videos

  • Utilized a generic protein tag for normalization of surface protein expression.
  • Employed fluorescence-activated cell sorting (FACS) for affinity-based cell sorting.
  • Mixed protein A variants with an 8-fold affinity difference at a 1:1000 ratio for sorting.

    • Main Results:

    • Successfully expressed four staphylococcal protein A variants with differing human IgG affinities on S. carnosus.
    • Demonstrated normalization of target-binding signals using a generic protein tag, mitigating expression biases.
    • Achieved approximately 140-fold enrichment of high-affinity binders from a 1:1000 mixture in a single FACS round.
    • Sorted the top 0.1% fraction based on the antigen binding to surface expression level ratio.

    Conclusions:

    • The developed staphylococcal surface display system shows potential for protein library selection applications.
    • The implemented normalization strategy enables fine affinity discrimination, critical for future library selections.
    • This system offers a robust platform for identifying high-affinity protein binders in combinatorial approaches.