Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants

• Published in: Plant physiology and biochemistry Vol. 108; pp. 139 - 144
• Main Authors: Abdoli Nasab, Maryam, Jalali Javaran, Mokhtar, Cusido, Rosa M., Palazon, Javier
• Format: Journal Article
• Language: English
• Published: France Elsevier Masson SAS 01.11.2016
• Subjects: Affinity chromatography; Chemical Fractionation; Chloroplasts - genetics; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; His-tag; Nicotiana - genetics; Nicotiana - metabolism; Plants, Genetically Modified; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Tissue Plasminogen Activator - genetics; Tissue Plasminogen Activator - isolation & purification; Tissue Plasminogen Activator - metabolism; tPA; Transplastomic plants; tPA; Affinity chromatography; Transplastomic plants; His-tag
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2016.06.029

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. •The recombinant tissue plasminogen activator (rtPA) protein was purified from transplastomic in active form tobacco plants using nickel resin and a chromatography column.•The protein was purified from total soluble protein and insoluble protein aggregates.•The chloroplasts were isolated from transplastomic plants and the protein was extracted from them.