Optimized isolation and purification of native glycoprotein B from herpes simplex virus 1: a streamlined approach
-
Mohammad Yasaghi
,
Ahad Yamchi
,
Alijan Tabarraei
,
Sara Salari
,
Abdolvahab Moradi
,
Seyedeh Delafruz Hosseini
Abstract
Background and Objectives: Viral membrane glycoproteins are essential for host cell recognition, membrane fusion and immune evasion, making them critical targets for antiviral therapies and vaccine development. However, their isolation in native conformation is challenging due to structural complexity and limitations of conventional purification methods. The aim of current study was to develop a cost-effective, reproducible method for the isolation and purification of glycoprotein B (gB) from Herpes Simplex Virus type 1 (HSV-1) while maintaining its native conformation for functional and interaction studies.
Materials and Methods: HSV-1 particles were concentrated via ultracentrifugation and membrane proteins were extracted using a modified protocol of the Mem-PER™ Plus Membrane Protein Extraction Kit. Native PAGE with a 4-8% gradient gel was employed to isolate multimeric gB (~300 kDa), followed by electroelution to extract the protein from the gel. The purity and integrity of gB were validated using SDS-PAGE and Western blot analysis.
Results: The method successfully isolated glycoprotein B in its native multimeric form with high purity and adequate concentration (0.157 mg/mL). The pH of the native gel (8.3) and the high molecular weight of gB facilitated separation from other viral surface proteins. SDS-PAGE and Western blot confirmed the specificity and structural integrity of the purified protein.
Conclusion: This study introduces a cost-effective and reliable method for isolating viral glycoproteins in their native conformation. The approach offers significant advantages over traditional chromatography-based techniques, making it ideal for research-scale applications, including functional and interaction studies.
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31. Lun C, Waheed AA, Majadly A, Powell N, Freed EO. Mechanism of viral Glycoprotein Targeting by Membrane-Associated RING-CH proteins. mBio 2021; 12(2): e00219-21.
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33. Zhu Z, Chen H, Ren J, Lu J, Gu C, Lynch K, et al. Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins. Talanta 2018; 179: 588-593.
34. Schwahn A, Baek J, Lin S, Pohl CA, Cook K. A Universal Eluent System for method Scouting and Separation of Biotherapeutic proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography. Anal Chem 2022; 94: 16369-16375.
35. Kadav P, Edwards J, Bandyopadhyay P, Dam T. Detection and purification of lectins and glycoproteins by a non‐column chromatographic technique. FASEB J 2021; 35: 10.1096/fasebj.2021.35.S1.04738.
36. Kusuma SAF, Fadhlillah M, Rostinawati T, Maisyarah IT, Syafitri RIP, Subroto T. Gel protein extraction’s impact on Conformational epitopes of Linear Non-Tagged MPT64 protein. Gels 2023; 9: 578.
37. Farmerie L, Rustandi RR, Loughney JW, Dawod M. Recent advances in isoelectric focusing of proteins and peptides. J Chromatogr A 2021; 1651: 462274.
2. Tang T, Bidon M, Jaimes JA, Whittaker GR, Daniel S. Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral Res 2020; 178: 104792.
3. Burnie J, Guzzo C. The Incorporation of Host Proteins into the External HIV-1 Envelope. Viruses 2019; 11: 85.
4. Seyedpour S, Khodaei B, Loghman AH, Seyedpour N, Kisomi MF, Balibegloo M, et al. Targeted therapy strategies against SARS‐CoV‐2 cell entry mechanisms: A systematic review of in vitro and in vivo studies. J Cell Physiol 2021; 236: 2364-2392.
5. Braun RJ, Kinkl N, Beer M, Ueffing M. Two-dimensional electrophoresis of membrane proteins. Anal
Bioanal Chem 2007; 389: 1033-1045.
6. Tao X, Zhao C, MacKinnon R. Membrane protein isolation and structure determination in cell-derived membrane vesicles. Proc Natl Acad Sci U S A 2023; 120(18): e2302325120.
7. Boxi A, Parikh I, S RB, S SK. Current Trends in Protein Purification: A Review. Int J Sci Res Sci Technol 2020; 7: 279-310.
8. Sabapathy T, Helmerhorst E, Bottomley S, Babaeff S, Munyard K, Newsholme P, et al. Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor. Biochim Biophys Acta Biomembr 2019; 1861: 1204-1212.
9. Majeed S, Ahmad AB, Adetuyi O, Evans EG, Stoll S, Georgieva ER. Protein engineering and biochemical/biophysical approaches for structural studies of small membrane proteins and their complexes: Application to viroporins. FASEB J 2022; 36: 10.1096/fasebj.2022.36.S1.R2569.
10. Gonzalez KJ, Huang J, Criado MF, Banerjee A, Tompkins SM, Mousa JJ, et al. A general computational design strategy for stabilizing viral class I fusion proteins. Nat Commun 2024; 15: 1335.
11. Oluwole A, Shutin D, Bolla JR. Mass spectrometry of intact membrane proteins: shifting towards a more native-like context. Essays Biochem 2023; 67: 201-213.
12. Grosche L, Döhner K, Düthorn A, Hickford-Martinez A, Steinkasserer A, Sodeik B. Herpes simplex Virus Type 1 propagation, Titration and Single-step growth Curves. Bio Protoc 2019; 9(23): e3441.
13. Qoronfleh MW, Benton B, Ignacio R, Kaboord B. Selective Enrichment of Membrane proteins by Partition phase Separation for Proteomic studies. J Biomed Biotechnol 2003; 2003: 249-255.
14. Kielkopf CL, Bauer W, Urbatsch IL. Sodium Dodecyl Sulfate-Polyacrylamide gel Electrophoresis of proteins. Cold Spring Harb Protoc 2021; 2021: 10.1101/pdb.prot102228.
15. Hashemi M, Aghamaali M, Madani R, Emami T. Purification of Nucleoprotein of H9N2 Avian Influenza Virus Strain by Electroelution. Iran Red Crescent Med J 2019; 21(10): e96170.
16. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al (2005). Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, Ed., The Proteomics Protocols Handbook, Humana Press, New York, 571-607. https://doi.org/10.1385/1-59259-890-0:571
17. Arndt C, Koristka S, Feldmann A, Bachmann M. Native Polyacrylamide Gels. Methods Mol Biol 2019; 1855: 87-91.
18. Wittig I, Braun HP, Schägger H. Blue native PAGE. Nat Protoc 2006; 1: 418-428.
19. Tang X, Pikal MJ. Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm Res 2004; 21: 191-200.
20. Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 1989; 182: 319-236.
21. Mahmood T, Yang P-C. Western blot: technique, theory, and trouble shooting. N Am J Med Sci 2012; 4: 429-434.
22. Komala Sari T, Gianopulos KA, Weed DJ, Schneider SM, Pritchard SM, Nicola AV. Herpes simplex virus Glycoprotein C Regulates Low-pH Entry. mSphere 2020; 5(1): e00826-19.
23. Perez-Romero P, Perez A, Capul A, Montgomery R, Fuller AO. Herpes simplex virus entry mediator associates in infected cells in a complex with viral proteins gD and at least gH. J Virol 2005; 79: 4540-4544.
24. Chouljenko DV, Kim IJ, Chouljenko VN, Subramanian R, Walker JD, Kousoulas KG. Functional Hierarchy of Herpes simplex virus 1 Viral Glycoproteins in cytoplasmic virion Envelopment and egress. J Virol 2012; 86: 4262-4270.
25. Feng T, Zhang J, Chen Z, Pan W, Chen Z, Yan YD, et al. Glycosylation of viral proteins: Implication in virus–host interaction and virulence. Virulence 2022; 13: 670-683.
26. Spear P. Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol 2004; 6: 401-410.
27. Kumari M, Tetala KKR. A review on recent advances in the enrichment of glycopeptides and glycoproteins by liquid chromatographic methods: 2016–Present. Electrophoresis 2022; 43: 388-402.
28. Salod Z, Mahomed O. Mapping potential vaccine Candidates Predicted by VaxiJen for different viral Pathogens between 2017–2021—A Scoping review. Vaccines (Basel) 2022; 10: 1785.
29. Azadfar S, Yamchi A, Majd A, Tabarraei A. Expression and purification of HCMV glycoprotein B full protein in Escherichia coli. J Complement Med Res 2020; 11: 60-67.
30. Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges Associated with the formation of Recombinant Protein inclusion bodies in Escherichia coli and Strategies to address them for industrial Applications. Front Bioeng Biotechnol 2021; 9: 630551.
31. Lun C, Waheed AA, Majadly A, Powell N, Freed EO. Mechanism of viral Glycoprotein Targeting by Membrane-Associated RING-CH proteins. mBio 2021; 12(2): e00219-21.
32. Lombardi L, Falanga A, Del Genio V, Palomba L, Galdiero M, Franci G, et al. A boost to the antiviral activity: Cholesterol tagged peptides derived from glycoprotein B of Herpes Simplex virus type I. Int J Biol Macromol 2020; 162: 882-893.
33. Zhu Z, Chen H, Ren J, Lu J, Gu C, Lynch K, et al. Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins. Talanta 2018; 179: 588-593.
34. Schwahn A, Baek J, Lin S, Pohl CA, Cook K. A Universal Eluent System for method Scouting and Separation of Biotherapeutic proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography. Anal Chem 2022; 94: 16369-16375.
35. Kadav P, Edwards J, Bandyopadhyay P, Dam T. Detection and purification of lectins and glycoproteins by a non‐column chromatographic technique. FASEB J 2021; 35: 10.1096/fasebj.2021.35.S1.04738.
36. Kusuma SAF, Fadhlillah M, Rostinawati T, Maisyarah IT, Syafitri RIP, Subroto T. Gel protein extraction’s impact on Conformational epitopes of Linear Non-Tagged MPT64 protein. Gels 2023; 9: 578.
37. Farmerie L, Rustandi RR, Loughney JW, Dawod M. Recent advances in isoelectric focusing of proteins and peptides. J Chromatogr A 2021; 1651: 462274.
| Files | ||
| Issue | Vol 17 No 3 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i3.18833 | |
| Keywords | ||
| Herpes simplex virus 1 Electroelution Native polyacrylamide gel electrophoresis Viral envelope proteins Protein purification | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Yasaghi M, Yamchi A, Tabarraei A, Salari S, Moradi A, Hosseini SD. Optimized isolation and purification of native glycoprotein B from herpes simplex virus 1: a streamlined approach. Iran J Microbiol. 2025;17(3):503-510.
