Amino Acids

pp 1–10 | Cite as

Site-specific derivatization of human interferon β-1a at lysine residues using microbial transglutaminase

  • Barbara Spolaore
  • Giacomo Forzato
  • Angelo Fontana
Original Article
  • 67 Downloads

Abstract

Microbial transglutaminase (TGase) has been successfully used to produce site-specific protein conjugates derivatized at the level of glutamine (Gln) or lysine (Lys) residues with diverse applications. Here, we study the drug human interferon β-1a (IFN) as a substrate of TGase. The derivatization reaction was performed using carbobenzoxy-l-glutaminyl-glycine to modify Lys residues and dansylcadaverine for Gln residues. The 166 amino acids polypeptide chain of IFN β-1a contains 11 Lys and 11 Gln residues potential sites of TGase derivatization. By means of mass spectrometry analyses, we demonstrate the highly selective derivatization of this protein by TGase at the level of Lys115 and as secondary site at the level of Lys33, while no reactive Gln residue was detected. Limited proteolysis experiments were performed on IFN to determine flexible regions of the protein under physiological conditions. Interestingly, primary and secondary sites of limited proteolysis and of TGase derivatization occur at the same regions of the polypeptide chain, indicating that the extraordinary selectivity of the TGase-mediated reaction is dictated by the conformational features of the protein substrate. We envisage that the TGase-mediated derivatization of IFN can be used to produce interesting derivatives of this important therapeutic protein.

Keywords

Human interferon β-1a Protein conjugation Transglutaminase Limited proteolysis 

Abbreviations

ACN

Acetonitrile

DC

Dansylcadaverine

E/S

Enzyme to substrate ratio

IFN

Human interferon β-1a

PEG

Polyethylene glycol

TFA

Trifluoroacetic acid

TGase

Transglutaminase

TCEP

Tris(2-carboxyethyl)phosphine

ZQG

Carbobenzoxy-l-glutaminyl-glycine

Notes

Acknowledgements

We acknowledge Silvia Gelio for conducting some experiments. This work was supported by the University of Padua (60A04-3887/12 and 60A04-8780/15).

Compliance with ethical standards

Conflicts of interest

The authors declare that there is no conflict of interest with regard to publication of this research work.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

726_2018_2563_MOESM1_ESM.docx (140 kb)
Supplementary material 1 (DOCX 140 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical and Pharmacological SciencesUniversity of PaduaPaduaItaly
  2. 2.CRIBI Biotechnology CentreUniversity of PaduaPaduaItaly

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