Recombinant production of human papillomavirus type 16 E6 and E7 vaccine antigens in Chlamydomonas reinhardtii.

Cervical cancer remains a major health challenge in low- and middle-income countries, where access to prevention and treatment is limited. Persistent infection with high-risk human papillomavirus (HPV), particularly HPV16, drives malignant transformation through the continuous expression of the viral oncoproteins E6 and E7. These tumor-specific antigens are promising targets for therapeutic vaccine development, but scalable and affordable production systems for recombinant HPV antigens are still lacking. This study establishes Chlamydomonas reinhardtii as a microalgal platform for producing HPV16 E6 and E7 proteins. Codon-optimized sequences encoding HPV16 E6 (519 bp) and E7 (481 bp) were integrated into the nuclear genome of C. reinhardtii using electroporation-mediated transformation. Protein analysis by SDS-PAGE and immunoblotting detected recombinant E6 (∼18 kDa) and E7 (∼11 kDa) at their predicted molecular weights, confirming successful translation. Critically, LC-MS/MS analysis identified multiple high-confidence tryptic peptides corresponding to both HPV antigens, with sequence coverage of 22-35%, providing definitive mass spectrometry-based validation of recombinant protein identity and integrity. These findings show that C. reinhardtii can serve as a scalable, platform for producing clinically relevant HPV16 antigens, supporting its potential application in the development of therapeutic vaccines for HPV-associated cancers.