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Related Experiment Videos

Multifunctional g3p-peptide tag for current phage display systems

C Beckmann1, B Haase, K N Timmis

  • 1Division of Microbiology, GBF-National Research Centre for Biotechnology, Braunschweig, Germany.

Journal of Immunological Methods
|July 22, 1998
PubMed
Summary

A new peptide tag derived from M13 phage gene-3 protein was identified and engineered into recombinant proteins. This tag enables sensitive detection and purification of single chain antibodies using monoclonal antibody 10C3.

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Area of Science:

  • Molecular Biology
  • Immunology
  • Biotechnology

Background:

  • Monoclonal antibody (mAb) 10C3 targets the gene-3 protein (g3p) of filamentous phage M13.
  • Previous work utilized mAb 10C3 to study g3p fusion protein expression in Escherichia coli and phage capsid incorporation.

Purpose of the Study:

  • To map the antigenic epitope of mAb 10C3 on the C-terminal half of g3p.
  • To engineer this epitope as a peptide tag for detecting and purifying recombinant proteins, including single chain antibodies.

Main Methods:

  • Epitope mapping using overlapping peptide sequences of g3p.
  • Introduction of the identified epitope as a tag into the phagemid pHEN1.
  • Detection of tagged single chain antibodies using ELISA and immunoblotting.
  • Determination of antibody affinity constant (K(D)) via surface plasmon resonance (SPR).

Related Experiment Videos

  • Single-step purification of recombinant proteins using immobilized mAb 10C3.

    • Main Results:

    • A minimal recognizable peptide epitope (11 amino acids, positions 292-302 of g3p) was identified.
    • Tagged single chain antibodies were detected at concentrations as low as 2 ng/mL (ELISA) and 4 ng/lane (immunoblot).
    • The antibody exhibited high affinity for the peptide tag (K(D) = 6.80 x 10(-10) M).
    • Recombinant proteins were successfully purified in one step using immobilized mAb 10C3.

    Conclusions:

    • The identified peptide tag and its corresponding mAb 10C3 provide a sensitive and versatile tool.
    • This system facilitates the detection and purification of recombinant proteins selected via phage display technology.