Protein chemical synthesis opens up new avenues for studying the biology and medicinal chemistry of synthetic proteins and provide new possibilities to generate biologics with new functional activities. Previously, we have reported that the unprotected peptide salicylaldehyde esters can chemoselectively ligate another unprotected peptide with N-terminal serine/threonine for Ser/Thr ligation (STL), or with N-terminal cysteine/penicillamine for Cys/Pen ligation (CPL). In the presentation, we will discuss the updated development of combining STL and CPL with protein-semisynthesis to synthesize large proteins. Furthermore, this strategy has enabled us to generate HMGB1 proteins with site-specific modifications for subsequent biochemical studies.