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Related Experiment Videos

Polyarginine as a multifunctional fusion tag.

Stephen M Fuchs1, Ronald T Raines

  • 1Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA. raines@biochem.wisc.edu.

Protein Science : a Publication of the Protein Society
|June 3, 2005
PubMed
Summary

Nonaarginine (R(9)) fusion tags enhance protein delivery into cells. While slightly reducing stability, the R(9) tag maintains enzyme activity and aids purification, proving versatile for protein applications.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Cationic peptides, like nonaarginine (R(9)), are utilized for delivering molecular cargo into mammalian cells.
  • Protein fusion tags can modify protein properties and facilitate applications.

Purpose of the Study:

  • To comprehensively analyze the impact of a nonaarginine (R(9)) tag on the biophysical and functional attributes of bovine pancreatic ribonuclease (RNase A).
  • To evaluate the utility of the R(9) tag for protein purification, immobilization, and cellular delivery.

Main Methods:

  • Fusion of R(9) tag to RNase A.
  • Differential scanning calorimetry to assess conformational stability.
  • Enzymatic activity assays.
  • Cation-exchange chromatography for purification.

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  • Immobilization on glass slides and silica resin.
  • Carboxypeptidase B treatment for tag removal.
  • Cellular uptake and cytotoxicity assays.

    • Main Results:

    • The R(9) tag decreased RNase A conformational stability (ΔT(m) = -8°C), an effect mitigated by salt.
    • Enzymatic activity of RNase A was preserved.
    • R(9) facilitated purification via cation-exchange chromatography and adsorption onto surfaces while retaining activity.
    • The tag was efficiently removed by carboxypeptidase B.
    • Cellular uptake and cytotoxicity of a modified RNase A (G88R RNase A) were significantly increased.

    Conclusions:

    • Polyarginine (R(9)) is a versatile protein fusion tag.
    • It enhances cellular uptake and cytotoxicity, aids in purification and immobilization, and can be precisely removed.
    • R(9) fusion holds promise for protein-based therapeutics and research tools.