Optimized isolation and purification of native glycoprotein B from herpes simplex virus 1: a streamlined approach
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 na- tive 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 con- centration (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 con- formation. The approach offers significant advantages over traditional chromatography-based techniques, making it ideal for research-scale applications, including functional and interaction studies.