title: 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.
authors.affiliation: Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Avenida Instituto Politécnico Nacional 2508, Colonia San Pedro Zacatenco, México Distrito Federal, CP, 07360.
authors.forename: Edgar García
authors.initials: EG
authors.lastname: López
authors.forename: Emma Gloria Ramos
authors.initials: EG
authors.lastname: Ramírez
authors.forename: Octavio Gómez
authors.initials: OG
authors.lastname: Gúzman
authors.forename: Graciano Calva
authors.initials: GC
authors.lastname: Calva
authors.forename: Armando
authors.initials: A
authors.lastname: Ariza-Castolo
authors.forename: Josefina
authors.initials: J
authors.lastname: Pérez-Vargas
authors.forename: Herminia Guadalupe Martínez
authors.initials: HG
authors.lastname: Rodríguez
mesh_terms: D019149:Bioreactors; D001937:Brassica / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019382:Human Growth Hormone / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018517:Plant Roots / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D046509:Tissue Culture Techniques / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone
keywords: Agrobacterium rhizogenes; GUSPlus; heterologous protein; pCAMBIA; somatotropin