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

## TL;DR

Scientists used transgenic tobacco plants to produce bioactive type III collagen, which could be useful for regenerative medicine.

## Contribution

This is the first report of triple-helical recombinant type III collagen production in transgenic tobacco plants.

## Key findings

- Transgenic tobacco plants coexpressing COL3A1 and modification enzymes produced thermally stable rhCOL3.
- Plant-derived rhCOL3 has a triple-helix structure and biological activity.
- Propeptides of rhCOL3 were correctly cleaved by coexpressed enzymes.

## Abstract

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 COL3A1, as well as a pMDV-COL3A1:5E vector that coexpressed COL3A1 and the enzymes required for its posttranslational modification. These two vectors were used to transform tobacco genetically. The COL3A1 gene was successfully coexpressed in tobacco plants with four enzymes that promote its posttranslational modification. The transcriptional level of COL3A1 in the transgenic lines coexpressing posttranslational modification genes was greater than that in the transgenic lines expressing only COL3A1. 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.