Transgenic Tobacco as a Bioreactor for the Production of Bioactive and Triple-Helical Recombinant Type III Collagen

Collagen is the primary protein in the extracellular matrix of human cells and the body and is essential for cell structure and function. Here, for the first time, we report a method for producing recombinant triple-helical collagen type III (rhCOL3) in transgenic tobacco as a bioreactor. We constructed a pMDV-COL3A1 vector containing the human type III collagen gene <i>COL3A1</i>, as well as a pMDV-COL3A1:5E vector that coexpressed <i>COL3A1</i> and the enzymes required for its posttranslational modification. These two vectors were used to transform tobacco genetically. The <i>COL3A1</i> gene was successfully coexpressed in tobacco plants with four enzymes that promote its posttranslational modification. The transcriptional level of <i>COL3A1</i> in the transgenic lines coexpressing posttranslational modification genes was greater than that in the transgenic lines expressing only <i>COL3A1</i>. The enzyme-modified recombinant collagen was subsequently purified from a COL3A1:5E transgenic line. Our experimental results demonstrated that the terminal propeptides of plant-derived rhCOL3 can be correctly cleaved through the enzymatic hydrolysis of procollagen by coexpressed procollagen C proteinase (PCP) and procollagen N proteinase (PNP). The plant-derived rhCOL3 was thermally stable because the purified peptide chains can form a triple helix structure. Experiments have shown that plant-derived rhCOL3 has biological activity. In this study, functional recombinant full-length mature type III collagen with a triple-helix structure was successfully expressed in tobacco, providing a foundational plant-made material for future applications of collagen in human skin and bone repair in regenerative medicine.