Stabilizing Peptide Fusion for Solving the Stability and Solubility Problems of Therapeutic Proteins

Purpose

Protein aggregation is a major stability problem of therapeutic proteins. We investigated whether a novel stabilizing peptide [acidic tail of synuclein (ATS) peptide] could be generally used to make a more stable and soluble form of therapeutic proteins, particularly those having solubility or aggregation problems.

Methods

We produced ATS fusion proteins by fusing the stabilizing peptide to three representative therapeutic proteins, and then compared the stress-induced aggregation profiles, thermostability, and solubility of them. We also compared the in vivo stability of these ATS fusion proteins by studying their pharmacokinetics in rats.

Results

The human growth hormone–ATS (hGH–ATS) and granulocyte colony-stimulating factor–ATS (G-CSF–ATS) fusion proteins were fully functional as determined by cell proliferation assay, and the ATS fusion proteins seemed to be very resistant to agitation, freeze/thaw, and heat stresses. The introduction of the ATS peptide significantly increased the storage and thermal stabilities of hGH and G-CSF. The human leptin–ATS fusion protein also seemed to be very resistant to aggregation induced by agitation, freeze/thaw, and heat stresses. Furthermore, the ATS peptide greatly increased the solubility of the fusion proteins. Finally, pharmacokinetic studies in rats revealed that the ATS fusion proteins are also more stable in vivo.

Conclusion

Our data demonstrate that a more stable and soluble form of therapeutic proteins can be produced by fusing the ATS peptide.

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Abbreviations

ATS:

acidic tail of synuclein

CD:

circular dichroism

G-CSF:

granulocyte colony-stimulating factor

GST:

glutathione S-transferase

hGH:

human growth hormone

Tm:

melting temperature

Tu:

temperature for the onset of unfolding

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Acknowledgments

We thank Dr. S.M. Park and K.J. Ahn for their technical assistance. This work was supported in part by a grant (R13-2002-054-02002-0) from the basic research program of the KOSEF.

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Correspondence to Jongsun Kim.

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E. N. Lee and Y. M. Kim equally contributed to this work.

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Lee, E.N., Kim, Y.M., Lee, H.J. et al. Stabilizing Peptide Fusion for Solving the Stability and Solubility Problems of Therapeutic Proteins. Pharm Res 22, 1735–1746 (2005). https://doi.org/10.1007/s11095-005-6489-4

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Key Words

  • protein aggregation
  • protein solubility
  • protein stability
  • stabilizing peptide
  • therapeutic proteins