High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3' splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 microg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter-signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. KEY POINTS: • The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 • The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins • The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity.