title: Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants.
abstract: Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis.
authors.affiliation: Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advanced Technology, P.O. Box 76315-117, Kerman, Iran. Electronic address: m.abdolinasab@kgut.ac.ir.
authors.forename: Maryam
authors.initials: M
authors.lastname: Abdoli Nasab
authors.affiliation: Department of Plant Breeding, Faculty of Agiculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran. Electronic address: m_jalali@modares.ac.ir.
authors.forename: Mokhtar
authors.initials: M
authors.lastname: Jalali Javaran
authors.affiliation: Department of Plant Physiology, Faculty of Pharmacy, University of Barcelona, Av. Joan, XXIII, S/n, 08028, Barcelona, Spain. Electronic address: rcusido@ub.edu.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Cusido
authors.affiliation: Department of Plant Physiology, Faculty of Pharmacy, University of Barcelona, Av. Joan, XXIII, S/n, 08028, Barcelona, Spain. Electronic address: javierpalazon@ub.edu.
authors.forename: Javier
authors.initials: J
authors.lastname: Palazon
mesh_terms: D005591:Chemical Fractionation; D002736:Chloroplasts / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator
keywords: Affinity chromatography; His-tag; Transplastomic plants; tPA