Coarse-grained molecular dynamics-guided immunoinformatics to explain the binder and non-binder classification of Cytotoxic T-cell epitope for SARS-CoV-2 peptide-based vaccine discovery.

Epitope-based peptide vaccine can elicit T-cell immunity against SARS-CoV-2 to clear the infection. However, finding the best epitope from the whole antigen is challenging. A peptide screening using immunoinformatics usually starts from MHC-binding peptide, immunogenicity, cross-reactivity with the human proteome, to toxicity analysis. This pipeline classified the peptides into three categories, i.e., strong-, weak-, and non-binder, without incorporating the structural aspect. For this reason, the molecular detail that discriminates the binders from non-binder is interesting to be investigated. In this study, five CTL epitopes against HLA-A*02:01 were identified from the coarse-grained molecular dynamics-guided immunoinformatics screening. The strong binder showed distinctive activities from the non-binder in terms of structural and energetic properties. Furthermore, the second residue from the nonameric peptide was most important in the interaction with HLA-A*02:01. By understanding the nature of MHC-peptide interaction, we hoped to improve the chance of finding the best epitope for a peptide vaccine candidate.

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity.

Human epidermal growth factor (hEGF) is a mitogenic protein widely used in pharmaceutical and cosmetic industries, thus recombinant DNA technology has been applied to meet the high demand for hEGF. The overexpression of recombinant protein in E. coli often leads to the formation of inclusion bodies (IBs). Mild solubilisation preserves the native secondary protein structure in IBs, thereby the high recovery of active protein from IBs. The redox system also plays a pivotal role in the formation of disulphide bonds during refolding of disulphide bond-containing protein. This study aimed to recover hEGF from bacterial IBs through freeze-thawing solubilisation and glutathione-based oxidative refolding. CBD-Ssp DnaB-hEGF fusion protein was expressed as IBs in E. coli, washed with Triton X-100 and urea to remove most protein contaminants, then the solubilised fusion protein was obtained by freeze-thawing with the addition of 2 M urea. The solubilised protein was subsequently refolded by intein cleavage via a glutathione-based redox system. The refolded hEGF demonstrated heat-resistant properties, interacted with specific antibodies on ELISA, stimulated keratinocyte proliferation and possessed significant in vivo wound healing properties on the 8th day, confirming that hEGF was correctly folded. In summary, the protocol described is suitable for the recovery of refolded hEGF from bacterial IBs by mild solubilisation and oxidative refolding.