Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

doi:10.1016/j.cjche.2021.09.027

Abstract

Abstract: Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix I, II, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid–solid interfaces. Among the three affinity dextran-coated Fe₃O₄ magnetic nanoparticles (Fe₃O₄@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix I (Fe₃O₄@Dx-Z₁ MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix II (Fe₃O₄@Dx-Z₂ MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe₃O₄@Dx-Z₁ MNPs because of less positive charged residues on Helix I (K6) than Helix II (R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G (IgG) binding was observed on Fe₃O₄@Dx-Z₁ MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe₃O₄@Dx-Z₁ MNPs. Finally, the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid–solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

Key words: Adsorption, Interface, Thermodynamics, Protein A ligand, Immobilization, Molecular structure

摘要： Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix I, II, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid–solid interfaces. Among the three affinity dextran-coated Fe₃O₄ magnetic nanoparticles (Fe₃O₄@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix I (Fe₃O₄@Dx-Z₁ MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix II (Fe₃O₄@Dx-Z₂ MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe₃O₄@Dx-Z₁ MNPs because of less positive charged residues on Helix I (K6) than Helix II (R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G (IgG) binding was observed on Fe₃O₄@Dx-Z₁ MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe₃O₄@Dx-Z₁ MNPs. Finally, the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid–solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

关键词: Adsorption, Interface, Thermodynamics, Protein A ligand, Immobilization, Molecular structure

Yi Shen, Xinshuang Chu, Qinghong Shi. Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis[J]. Chinese Journal of Chemical Engineering, 2023, 54(2): 232-239.

Yi Shen, Xinshuang Chu, Qinghong Shi. Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis[J]. 中国化学工程学报, 2023, 54(2): 232-239.

References

[1] D.J. Bornhop, J.C. Latham, A. Kussrow, D.A. Markov, R.D. Jones, H.S. Sørensen, Free-solution, label-free molecular interactions studied by back-scattering interferometry, Science 317 (5845) (2007) 1732–1736.
[2] K.P.S. Dancil, D.P. Greiner, M.J. Sailor, A porous silicon optical biosensor: Detection of reversible binding of IgG to a protein A-modified surface, J. Am. Chem. Soc. 121 (34) (1999) 7925–7930.
[3] S. Ghose, M. Allen, B. Hubbard, C. Brooks, S.M. Cramer, Antibody variable region interactions with Protein A: Implications for the development of generic purification processes, Biotechnol Bioeng 92 (6) (2005) 665–673.
[4] J.S. Seo, S. Lee, C.D. Poulter, Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays, J Am Chem Soc 135 (24) (2013) 8973–8980.
[5] T. Tanaka, T. Matsunaga, Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes, Anal Chem 72 (15) (2000) 3518–3522.
[6] J. Turková, Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function, J Chromatogr B Biomed Sci Appl 722 (1–2) (1999) 11–31.
[7] N. Tajima, M. Takai, K. Ishihara, Significance of antibody orientation unraveled: Well-oriented antibodies recorded high binding affinity, Anal Chem 83 (6) (2011) 1969–1976.
[8] T. Ikeda, Y. Hata, K. Ninomiya, Y. Ikura, K. Takeguchi, S. Aoyagi, R. Hirota, A. Kuroda, Oriented immobilization of antibodies on a silicon wafer using Si-tagged protein A, Anal Biochem 385 (1) (2009) 132–137.
[9] H. Wang, Y.L. Liu, Y.H. Yang, T. Deng, G.L. Shen, R.Q. Yu, A protein A-based orientation-controlled immobilization strategy for antibodies using nanometer-sized gold particles and plasma-polymerized film, Anal Biochem 324 (2) (2004) 219–226.
[10] M. Freiherr von Roman, S. Berensmeier, Improving the binding capacities of protein A chromatographic materials by means of ligand polymerization, J Chromatogr A 1347 (2014) 80–86.
[11] A. Makaraviciute, A. Ramanaviciene, Site-directed antibody immobilization techniques for immunosensors, Biosens Bioelectron 50 (2013) 460–471.
[12] Z.Q. Chen, X.S. Wang, X. Cheng, W.J. Yang, Y.N. Wu, F.F. Fu, Specifically and visually detect methyl-mercury and ethyl-mercury in fish sample based on DNA-templated alloy Ag-Au nanoparticles, Anal Chem 90 (8) (2018) 5489–5495.
[13] X.H. Yang, L.M. Huan, X.S. Chu, Y. Sun, Q.H. Shi, A comparative investigation of random and oriented immobilization of protein A ligands on the binding of immunoglobulin G, Biochem. Eng. J. 139 (2018) 15–24.
[14] G. Anand, S. Sharma, A.K. Dutta, S.K. Kumar, G. Belfort, Conformational transitions of adsorbed proteins on surfaces of varying polarity, Langmuir 26 (13) (2010) 10803–10811.
[15] T.A. Horbett, J.L. Brash, Proteins at Interfaces II: Fundamental and Applications, American Chemical Society, Washington, DC, 1995.
[16] C.V. Kumar, A. Chaudhari, Proteins immobilized at the galleries of layered α-zirconium phosphate: Structure and activity studies, J. Am. Chem. Soc. 122 (5) (2000) 830–837.
[17] Shen Z, Yan H, Zhang Y, Mernaugh RL, Zeng X, Engineering peptide linkers for scFv immunosensors, Anal Chem 80 (6) (2008) 1910–1917.
[18] A.M. Klibanov, Enzyme stabilization by immobilization, Anal Biochem 93 (1) (1979) 1–25.
[19] S. Ghose, B. Hubbard, S.M. Cramer, Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials, Biotechnol Bioeng 96 (4) (2007) 768–779.
[20] J. Kikuchi, Y. Mitsui, T. Asakura, K. Hasuda, H. Araki, K. Owaku, Spectroscopic investigation of tertiary fold of staphylococcal protein A to explore its engineering application, Biomaterials 20 (7) (1999) 647–654.
[21] Z. Wang, Y. Shen, Q.H. Shi, Y. Sun, Insights into the molecular structure of immobilized protein A ligands on dextran-coated nanoparticles: Comprehensive spectroscopic investigation, Biochem. Eng. J. 146 (2019) 20–30.
[22] K. Can, M. Ozmen, M. Ersoz, Immobilization of albumin on aminosilane modified superparamagnetic magnetite nanoparticles and its characterization, Colloids Surf B Biointerfaces 71 (1) (2009) 154–159.
[23] A. Mukhopadhyay, N. Joshi, K. Chattopadhyay, G. De, A facile synthesis of PEG-coated magnetite (Fe₃O₄) nanoparticles and their prevention of the reduction of cytochrome C, ACS Appl Mater Interfaces 4 (1) (2012) 142–149.
[24] A.M. Predescu, E. Matei, A.C. Berbecaru, C. Pantilimon, C. Drăgan, R. Vidu, C. Predescu, V. Kuncser, Synthesis and characterization of dextran-coated iron oxide nanoparticles, R Soc Open Sci 5 (3) (2018) 171525.
[25] C. Zhang, X.M. Shi, F.F. Yu, Y. Quan, Preparation of dummy molecularly imprinted polymers based on dextran-modified magnetic nanoparticles Fe₃O₄ for the selective detection of acrylamide in potato chips, Food Chem 317 (2020) 126431.
[26] Xu H, Cui L, Tong N, Gu H, Development of high magnetization Fe₃O₄/polystyrene/silica nanospheres via combined miniemulsion/emulsion polymerization, J Am Chem Soc 128 (49) (2006) 15582–15583.
[27] S.X. Zhang, Y.Y. Zhang, G.M. Bi, J.S. Liu, Z.G. Wang, Q. Xu, H. Xu, X.Y. Li, Mussel-inspired polydopamine biopolymer decorated with magnetic nanoparticles for multiple pollutants removal, J Hazard Mater 270 (2014) 27–34.
[28] B. Feng, R.Y. Hong, L.S. Wang, L. Guo, H.Z. Li, J. Ding, Y. Zheng, D.G. Wei, Synthesis of Fe₃O₄/APTES/PEG diacid functionalized magnetic nanoparticles for MR imaging, Colloids Surfaces A: Physicochem. Eng. Aspects 328 (1–3) (2008) 52–59.
[29] A. Bhambhani, C.V. Kumar, Tuning the properties of Hb intercalated in the galleries of α-ZrP with ionic strength: Improved structure retention and enhanced activity, Chem. Mater. 18 (3) (2006) 740–747.
[30] R. Chowdhury, B. Stromer, B. Pokharel, C.V. Kumar, Control of enzyme-solid interactions via chemical modification, Langmuir 28 (32) (2012) 11881–11889.
[31] A.C. Braisted, J.A. Wells, Minimizing a binding domain from protein A, Proc Natl Acad Sci USA 93 (12) (1996) 5688–5692.
[32] A. Micsonai, F. Wien, É. Bulyáki, J. Kun, É. Moussong, Y.H. Lee, Y. Goto, M. Réfrégiers, J. Kardos, BeStSel: A web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra, Nucleic Acids Res 46 (W1) (2018) W315–W322.
[33] A. Micsonai, F. Wien, L. Kernya, Y.H. Lee, Y. Goto, M. Réfrégiers, J. Kardos, Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy, Proc Natl Acad Sci USA 112 (24) (2015) E3095–E3103.
[34] L.N. Lund, T. Christensen, E. Toone, G. Houen, A. Staby, P.M. St Hilaire, Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry, J Mol Recognit 24 (6) (2011) 945–952.
[35] M.A. Starovasnik, M.P. O'Connell, W.J. Fairbrother, R.F. Kelley, Antibody variable region binding by Staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain, Protein Sci 8 (7) (1999) 1423–1431.
[36] H. Nagai, K. Kuwabara, G. Carta, Temperature dependence of the dissociation constants of several amino acids, J. Chem. Eng. Data 53 (3) (2008) 619–627.
[37] H.M. Yang, R.M. Bao, Y.Z. Cheng, J.B. Tang, Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: an efficient platform for 3D IgG immobilization, Anal Chim Acta 872 (2015) 1–6.
[38] H.O. Yang, P.V. Gurgel, R.G. Carbonell, Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography, J Chromatogr A 1216 (6) (2009) 910–918.