Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector.

OBJECTIVE: To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants. RESULTS: The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml. CONCLUSIONS: The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry.

Production of human tissue plasminogen activator (tPA) in Cucumis sativus.

Tissue plasminogen activator (tPA) as a serine protease with 72 kD molecular mass and 527 amino acids plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The collective production of this drug in plants such as cucumber, one of the most important vegetables in the world, could reduce its production costs. In this study, after scrutiny of the appropriate regeneration of cucumber plant (Isfahan variety) on MS medium with naphthalene acetic acid hormone (NAA; 0/1 mg L⁻¹) and benzyl amino purine hormone (BAP; 3 mg L⁻¹) hormones, the cloned human tPA gene under the CaMV 35S promoter and NOS terminator into pBI121 plasmid was transferred into cotyledon explants by Agrobacterium tumefaciens strain LBA4404. Subsequent to the regeneration of inoculated explants on the selective medium, the persistence of tPA gene in recombinant plants was confirmed by polymerase chain reaction (PCR) with specific primers. To evaluate the tPA gene expression in transgenic plants, RNA was extracted and the tPA gene transcription was confirmed by reverse-transcription (RT) PCR. Followed the extraction of protein from the leaves of transgenic plants, the presence of tPA protein was confirmed by dot blot and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis in order to survey the production of recombinant tPA protein. The enzyme-linked immunosorbent assay (ELISA) test was used for recombinant tPA protein level in transgenic cucumber plants. It was counted between 0.8 and 1%, and based on this, it was concluded that the presence of three expressions of regulatory factors (CaMV 35S, Kozak, NOS) and KDEL signal in the construct caused the increase of the tPA gene expression in cucumber plants.

Expression analysis and purification of human recombinant tissue type plasminogen activator (rt-PA) from transgenic tobacco plants.

Recombinant tissue-type plasminogen activator (rt-PA) has been produced in different hosts. In this research, transgenic tobacco was selected for production of human rt-PA. Transgenic plants were analyzed by polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. The protein was extracted by Lysine Sepharose chromatography column and was further purified by HiTrap desalting column. The function of eluted protein was analyzed on zymography gel. The results showed that the 1.7-kb cDNA of tissue-type plasminogen activator (t-PA) (as well as a shortened 650-bp transcript of t-PA) has been expressed in transgenic plants. The anticipated 63-kD protein band and an additional 53-kD protein were observed in transgenic plants. Finally, zymography assay revealed that the purified rt-PA has anticipated appropriate activity comparable to a positive control drug (Alteplase). On the whole, we can say that transgenic tobacco is a good alternative host for production of t-PA.

Abscisic acid regulates gene expression in cortical fiber cells and silica cells of rice shoots.

Drought-induced growth arrest is a major cause of yield loss in crops and is mediated in part by abscisic acid (ABA). The aim of this study was to identify the cell types targeted by ABA during arrest. As transcription factors ABI3 and ABI5 are essential for ABA-induced growth arrest in Arabidopsis, blast was used to identify OsVP1 and OsABF1 as their structural orthologues in rice (Oryza sativa), and employed RNA in situ hybridization to reveal the cell types accumulating the corresponding transcripts in response to ABA. Exogenous ABA arrested the growth of leaves 1, 2 and 3 in young rice shoots and inhibited secondary cell-wall formation in sclerenchyma, including expression of the cellulose synthase gene OsCesA9. Transcripts for OsVP1, OsABF1 and of the putative target genes OsEm, OsLEA3 and WSI18, increased under ABA, accumulating principally in the cytosol of the major support cells (sclerenchymatous cortical fiber cells and epidermal silica cells) of slowly growing leaf 1. Rapidly growing immature tissues in leaves 2 and 3 accumulated OsABF1, OsEm and WSI18 transcripts in the nuclei of all cells, irrespective of ABA treatment. It is concluded that during arrest of leaf growth, ABA targets support cells in maturing tissues. Target cells in immature tissues remain to be identified.

Transient expression of anti-VEFGR2 nanobody in Nicotiana tabacum and N. benthamiana.

In human, the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) is critical for tumor angiogenesis. This is a vital process for cancer tumor growth and metastasis. Blocking VEGF/VEGFR2 conjugation by antibodies inhibits the neovascularization and tumor metastasis. This investigation designed to use a transient expression platform for production of recombinant anti-VEGFR2 nanobody in tobacco plants. At first, anti-VEGFR2-specific nanobody gene was cloned in a Turnip mosaic virus (TuMV)-based vector, and then, it was expressed in Nicotiana benthamiana and Nicotiana tabacum cv. Xanthi transiently. The expression of nanobody in tobacco plants were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), dot blot, enzyme-linked immunosorbent assays (ELISA), and Western blot analysis. It was shown that tobacco plants could accumulate nanobody up to level 0.45% of total soluble protein (8.3 µg/100 mg of fresh leaf). This is the first report of the successful expression of the camelied anti-VEFGR2 nanobody gene in tobacco plants using a plant viral vector. This system provides a fast solution for production of pharmaceutical and commercial proteins such as anti-cancer nanobodies in tobacco plants.

Expression and large-scale production of human tissue plasminogen activator (t-PA) in transgenic tobacco plants using different signal peptides.

An attempt was made to assess the expression level and targeting of a human protein entitled recombinant tissue plasminogen activator (rt-PA) through accumulation in three cellular compartments including the endoplasmic reticulum and cytosolic and apoplastic spaces in transgenic tobacco plants. In this context, three chimeric constructs pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA were employed and transferred into the tobacco plants through a popular transformation-based system called Agrobacterium tumefaciens. As an initial screening system, the incorporation of the rt-PA gene in the genomic DNA of tobacco transgenic plants and the possible existence of the rt-PA-specific transcript in the total RNAs of transgenic plant leaves were confirmed via PCR and reverse transcription (RT)-PCR, respectively. Southern blot analysis, in addition, was used to determine the copy number of the corresponding gene (i.e., t-PA) transformed into the each transgenic plant; one or more copies were detected regarding transformants derived from all three abovementioned constructs. According to the enzyme-linked immunosorbent assay, the mean values of t-PA expression were calculated as 0.50, 0.68, and 0.69 µg/mg of the total soluble protein when a collection containing 30 transgenic plants transformed with pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA was taken into account, respectively. The zymography assay was lastly performed and concluded the expression of the properly folded rt-PA in this expression system. Our results, altogether, revealed that tobacco plants could be utilized as a bioreactor system for the large-scale production of enzymatically active t-PA and presumably other therapeutic recombinant proteins in large quantities.