MALDI‐TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor

Abstract

Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF-MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins.

Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors

“…It also has a lower operating cost than other types of bioreactors and has the advantage of easy scale-up for large-scale plant cell culture (Huang and McDonald 2012 ). Because of these advantages, López et al ( 2014 ) and Corbin et al ( 2016 ) used bioreactor cultures for recombinant protein production. However, there have been no reports on the use of bioreactors for the production of sweet proteins, such as miraculin.…”

Bacteria – Plant Interaction: Transgenic Roots for Human Health

“…Brassica oleracea var. Italica hairy roots carried pCAMBIA1105.1 binary vector accumulated isoform 1 of the human growth hormone (hGH1) in case of cultivation in 1.5-Litter mesh airlift bioreactor (López et al, 2014). The content of hGH1 7.8 ± 0.3 μg/g DW was for the bioreactor.…”

Plant Platforms for Efficient Heterologous Protein Production

“…An isoform of human growth hormone was produced in Brassica oleracea hairy root cultures. The B. rapa hairy root cultures showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility and highlighting the high homogeneity of the production system [180,181]. Although the recombinant proteins are accumulated or secreted by hairy root cultures in large quantities, proteins are degraded by the proteolytic activity of peptidases.…”