Expression and characterization of intein-cyclized trimer of staphylococcus aureus protein A domain Z

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dc.contributor.authorNandy, Suman
dc.contributor.authorMaranholkar, Vijay M.
dc.contributor.authorCrum, Mary
dc.contributor.authorWasden, Katherine
dc.contributor.authorPatil, Ujwal
dc.contributor.authorGoyal, Atul
dc.contributor.authorVu, Binh
dc.contributor.authorKourentzi, Katerina
dc.contributor.authorMo, William
dc.contributor.authorHenrickson, Amy
dc.contributor.authorDemeler, Borries
dc.contributor.authorSen, Mehmet
dc.contributor.authorWillson, Richard C.
dc.date.accessioned2024-08-19T17:30:06Z
dc.date.available2024-08-19T17:30:06Z
dc.date.issued2023
dc.descriptionOpen access article. Creative Commons Attribution 4.0 International license (CC BY 4.0) applies
dc.description.abstractStaphylococcus 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.
dc.description.peer-reviewYes
dc.identifier.citationNandy, S., Maranholkar, V. M., Crum, M., Wasden, K., Patil, U., Goyal, A., Vu, B., Kourentzi, K., Mo, W., Henrickson, A., Demeler, B., Sen, M., & Willson, R. C. (2023). Expression and characterization of intein-cyclized trimer of staphylococcus aureus protein A domain Z. International Journal of Molecular Sciences, 24(2), Article 1281. https://doi.org/10.3390/ijms24021281
dc.identifier.urihttps://hdl.handle.net/10133/6864
dc.language.isoen
dc.publisherMDPI
dc.publisher.departmentDepartment of Chemistry and Biochemistry
dc.publisher.facultyArts and Science
dc.publisher.institutionUniversity of Houston
dc.publisher.institutionUniversity of Lethbridge
dc.publisher.institutionUniversity of Montana
dc.publisher.institutionTecnologico de Monterrey
dc.publisher.urlhttps://doi.org/10.3390/ijms24021281
dc.subjectProtein A
dc.subjectCyclic Z3
dc.subjectSICLOPPS
dc.subjectTandem mass spectrometry
dc.subjectITC
dc.subjectDSF
dc.subjectSPR
dc.subjectCyclization
dc.subjectLigand
dc.subject.lcshProtein engineering
dc.subject.lcshLigands
dc.subject.lcshRing formation (Chemistry)
dc.titleExpression and characterization of intein-cyclized trimer of staphylococcus aureus protein A domain Z
dc.typeArticle
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