Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z

Suman Nandy, Vijay M. Maranholkar, Mary Crum, Katherine Wasden, Ujwal Patil, Atul Goyal, Binh Vu, Katerina Kourentzi, William Mo, Amy Henrickson, Borries Demeler, Mehmet Sen, Richard C. Willson

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of −33.0 kcal/mol and −32.7 kcal/mol, and −T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Original languageEnglish
Article number1281
JournalInternational Journal of Molecular Sciences
Issue number2
StatePublished - Jan 9 2023


  • DSF
  • ITC
  • SPR
  • cyclic Z3
  • protein A
  • tandem mass spectrometry
  • Calorimetry/methods
  • Humans
  • Immunoglobulin G
  • Thermodynamics
  • Inteins/genetics
  • Protein Binding
  • Ligands
  • Staphylococcus aureus


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