Chlamydomonas reinhardtii for recombinant somatotropin production: an alternative expression vector

The human growth hormone (hGH) is a therapeutic protein widely used in medicine for treating growth disorders and metabolic deficiencies. In this study, we aimed to express recombinant human growth hormone (rhGH) in the chloroplast of Chlamydomonas reinhardtii , using the endogenous psbD promoter to regulate expression. The optimized hGH gene fused to a FLAG tag was cloned into the GH-psbH expression vector, which was integrated into the chloroplast genome through homologous recombination at the psbH locus. Transformed colonies resistant to kanamycin were screened by PCR, and homoplasmic lines were confirmed. Western blotting revealed the synthesis of a recombinant protein with a molecular weight of approximately 23.6 ± 0.3 kDa, consistent with the predicted size of rhGH-FLAG. The best-performing clone showed a 4.5-fold higher accumulation compared to the reference colony. The recombinant protein was successfully purified by affinity chromatography, yielding an estimated concentration of 1.33 mg/L. These results demonstrate the effectiveness of the psbD promoter in driving heterologous protein expression in C. reinhardtii chloroplasts, maintaining photosynthetic capacity and achieving higher accumulation levels than previously reported promoters. This study supports C. reinhardtii as a sustainable and promising platform to produce human therapeutic proteins.