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Related Experiment Videos

Enhancing protein expression in single HEK 293 cells.

Zhen Huang1, Gang Li, Weimin Pei

  • 1Department of Chemistry, Center for Neuroscience Research, University at Albany, SUNY, Albany, NY 12222, USA.

Journal of Neuroscience Methods
|January 18, 2005
PubMed
Summary

Coexpressing simian virus 40 large T antigen (TAg) with recombinant proteins significantly boosts expression levels in single human embryonic kidney (HEK 293) cells. This method enhances protein characterization at the single-cell level without affecting protein function.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Background:

  • Recombinant protein expression in heterologous systems like HEK 293 cells is crucial for research.
  • Efficient expression is vital for single-cell level protein characterization.
  • Current methods may lack sufficient efficiency for certain applications.

Purpose of the Study:

  • To develop a simple method for enhancing recombinant protein expression efficiency in single HEK 293 cells.
  • To investigate the effect of coexpressing simian virus 40 large T antigen (TAg) on protein expression.
  • To validate the method for both membrane and soluble proteins and assess functional impact.

Main Methods:

  • Coexpression of simian virus 40 large T antigen (TAg) with target proteins in HEK 293 cells.

Related Experiment Videos

  • Optimization of the TAg to target protein plasmid ratio (found to be 1:10).
  • Assessment of protein expression using the GluR2 receptor and green fluorescent protein (GFP) as models.
  • Functional assays to determine the impact of TAg on receptor activity.

    • Main Results:

    • Coexpression of TAg increased GluR2 receptor expression approximately seven-fold in single HEK 293S cells.
    • Fluorescence imaging confirmed a similar increase in GFP expression upon TAg coexpression.
    • The optimal TAg:receptor plasmid ratio was determined to be 1:10.
    • Protein function, exemplified by the GluR2 receptor, remained unaffected by TAg presence.

    Conclusions:

    • Coexpression of TAg is a simple and effective method to enhance recombinant protein expression in single HEK 293 cells.
    • This technique is applicable to both membrane and soluble proteins, facilitating single-cell level studies.
    • The method allows for the direct study of protein function in intact cells, particularly beneficial for membrane proteins.