Expression and Large-Scale Production of Human

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Plants Using Different Signal Peptides. Hojjat Ghasemi Goojani & Mokhtar Jalali Javaran &. Jaber Nasiri & Esmaeel Ghasemi Goojani &. Houshang Alizadeh.
Appl Biochem Biotechnol DOI 10.1007/s12010-013-0115-4

Expression and Large-Scale Production of Human Tissue Plasminogen Activator (t-PA) in Transgenic Tobacco Plants Using Different Signal Peptides Hojjat Ghasemi Goojani & Mokhtar Jalali Javaran & Jaber Nasiri & Esmaeel Ghasemi Goojani & Houshang Alizadeh

Received: 27 August 2012 / Accepted: 14 January 2013 # Springer Science+Business Media New York 2013

Abstract 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-PAspecific transcript in the total RNAs of transgenic plant leaves were confirmed via PCR and reverse transcription (RT)-PCR, respectively. Southern blot analysis, in addition, H. G. Goojani : M. J. Javaran Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran H. G. Goojani e-mail: [email protected] M. J. Javaran e-mail: [email protected] J. Nasiri Department of Agronomy and Plant Breeding, Division of Molecular Plant Genetics, University of Tehran, Karaj, Tehran, Iran e-mail: [email protected] E. G. Goojani Department of Agronomy and Plant Breeding, Agricultural College, University of Tehran, Karaj, Iran e-mail: [email protected] H. Alizadeh (*) Department of Agricultural Biotechnology, Agricultural College, University of Tehran, Karaj, Iran e-mail: [email protected]

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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 pBIExt-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. Keywords Agrobacterium tumefaciens . Bioreactor system . Transgenic tobacco . Human tissue plasminogen activator

Introduction Regardless of the immense existence of conventional expression systems, either prokaryotic organisms including Escherichia coli or eukaryotic ones (i.e., fungal, insect, and mammalian cells), due in part to several remarkable drawbacks of the aforementioned approaches, other reliable scenarios including plants have been preferred to be utilized widely (as reviewed in [1]). For instance, while bacteria are an inexpensive and appropriate production system [1], the eukaryotic proteins produced via such organisms may not have the desired biological activity or stability [2], high equipment and production costs are required, probable contamination with pathogens may occur [3], and lastly, the final product possibly will contaminate via some bacterial compounds that are toxic or, in some cases, increase body temperature in both humans and animals (pyrogens) [2]. On the contrary, over the last 20 years, the existence of a number of unique features of plantbased expression systems has resulted in a broad shift of attention of scientists towards developing transgenic plants worldwide regarding large-scale production of industrial enzymes and pharmaceutical proteins as well [1, 2, 4–6]. Briefly, transgenic plants (due to their noteworthy characteristics including rapid scalability; the ability to correctly fold, assemble, and process complex proteins; the potential for direct oral administration of unprocessed or partially processed plant material [7, 8]; no risk of contamination with human pathogens; feasibility to expand; and the ability to carry out proper post-translational modifications, e.g., accurate glycosylation, which plays a pivotal role for the biological activity and stability of human therapeutic proteins) have accordingly shown great potential to offer a safe, efficient, and cost-effective means in manufacturing a great deal of recombinant pharmaceutical proteins worldwide, surprisingly in a cost-effective manner [2, 7, 9, 10]. Albeit a number of plant host systems have been proposed as reliable bioreactors to produce heterologous proteins, among which, the tobacco plant (Nicotiana tabacum L.) has been proven historically as the most established ones in yielding recombinant protein(s) [3, 6, 11]. The plant, in fact, has a high biomass yield (more than 100,000 kg per ha), and the platform is based on leaves [12], eliminating the need for flowering and thus minimizing the contingency of gene escape into the environment through pollen or seed dispersion [6]. Likewise, in view of the fact that tobacco is a non-food and non-feed crop, there is accordingly little risk of transgenic material contaminating the food chain [13, 14].

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The fibrinolytic system consists of an inactive pro-enzyme, plasminogen, which can be converted to the active enzyme, plasmin, which in turn degrades fibrin into soluble fibrin degradation products [15]. Plasminogen activators are enzymes that are used commonly in the treatment of cardiovascular and cerebrovascular obstructions resulting from heart attacks and strokes [16]. Two physiological plasminogen activators have been generally identified: the tissue-type activator (t-PA) and the urokinase-type plasminogen activator (u-PA) [17]. The enzyme tissue plasminogen activator (t-PA) is a multi-domain serine protease with 17 disulfide bonds, three main N-glycosylation and one O-glycosylation sites, and a molecular weight of approximately 68 kDa, which is medically useful for the dissolution of blood clots [2]. The enzyme is encoded by a 1,681-bp gene and consists of five distinct functional domains: a finger-binding domain, an epidermal growth factor domain, two kringle structures, and a catalytic domain [18, 19]. Nowadays, a mammalian cell culture system such as CHO cells is still utilized for the production of t-PA for commercial purposes [15], while E. coli is used for clinical purposes [20– 23]. Such popular procedures, nonetheless, are very costly; the total production costs have been measured to be more than US$2,000 per dose (100 mg) [24]. To remedy such considerable limitations, more recently, molecular farming as a cost-effective, safe, and large-scale system to produce a number of biopharmaceuticals and plant antibodies has undergone a broad spread worldwide including t-PA production (reviewed in [25, 26]). According to our knowledge, just a few well-documented investigations reporting on the expression of recombinant tissue plasminogen activator (rt-PA) in a plant system are available. Hahn et al. [15], for instance, were the pioneer for the successful production of this medically important human protein in tobacco plants. Similar studies were later carried out either using the same plant system [27, 28] or hairy roots of Oriental melon (Cucumis melo) [29]. In addition to the plant system, in an effort made by Nazari and Davoudi [19], the expression of t-PA was assessed by means of a system called Leishmania tarentolae. Interestingly, in the last study, replacement of the human signal sequence tPA with the signal sequence derived from Leishmania increased the secretion of the recombinant protein up to 30 times. The primary objective of the current investigation was to assess the use of signal targeting for the delivery of recombinant t-PA to some cell compartments including the endoplasmic reticulum (ER) (using KDEL), apoplastic space (using extensin signal [30]), and finally the cytosolic space (using SP signal belonging to the first part of the Zera signal), consequent to the accumulation of rt-PA inside of the cell wall [31]. Further, the expression level of rt-PA in different organelles was also scrutinized for the first time in a famous plant-based system called tobacco.

Materials and Methods Construction of Plasmids for Expression of t-PA in Different Compartments To generate the first construct called pBI-SP-tPA, first of all, a previously produced construct in our genomic lab (Alizadeh, unpublished data) namely pBI-SP-GR, containing Griffithsin gene as well as SP signal, was employed. Subsequent to the excision of the sequence encoding Griffithsin using EcoRI and XhoI restriction enzymes, a fragment encoding the t-PA gene (1,686 bp) with EcoRI/XhoI was cloned into the pBI-SP construct, using the same restriction sites (Fig. 1). Notably, the SP signal, belonging to the first part of the Zera signal, was included in the protein-targeting construct in order to produce rt-PA in the cytosolic space.

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Fig. 1 Schematic representation of the three different constructs used in current study. The a first, b second, and c third constructs on the basis of three different signals including KDEL signal (as pBI-KDEL-tPA construct), SP signal (as pBI-SP-tPA construct), and lastly extensin signal (as pBI-Ext-tPA construct), respectively

The second construct used in the current study was generated nearly similar to the first one; in fact, here, a previously produced construct in our genomic lab (Alizadeh, unpublished data) namely pBI-KDEL-GR containing both KDEL signal plus Griffithsin gene was utilized. Contrary to the first construct, the sequence encoding Griffithsin was cleaved using BamHI/XhoI. A fragment encoding t-PA and Kozak sequences with BamHI/XhoI was subsequently cloned into the same place occupied previously by the Griffithsin gene; the third construct (pBI-KDEL-tPA) was eventually created and utilized for further analyses (Fig. 1). The KDEL signal was introduced into the second construct to verify the successful expression of rt-PA in the ER. The methodology of generating the third construct followed those employed to fabricate the second one except that here the Griffithsin gene reside in our previously produced pBIEX-GR (also containing a genomic sequence encoding the extensin signal (GeneBank accession no. X02873)) was removed via BamHI/XhoI, superseded then by a genomic fragment encoding t-PA sequences (Fig. 1). Extensin signal was introduced into the third construct to verify the successful expression of rt-PA in the apoplastic space. Plant Transformation Small leaf pieces of tobacco plants (N. tabacum cv. Xathi), previously grown under controlled conditions, were utilized for Agrobacterium-mediated transformation (Agrobacterium tumefaciens strain LBA4404) followed by three cassettes including pBI-Ext-PA, pBI-SP-t-PA, and pBI-KDEL-t-PA. The transformation process of tobacco leaf discs was carried out according to the protocol manual of Horsch et al. [32]. Murashige and Skoog-based medium containing 2-naphthaleneacetic acid (0.1 mgL−1) and 6-benzylaminopurine (3 mgL−1), together with a selective medium of both kanamycin (100 mgL−1) and cefotaxime (200 mgL−1), was also employed to distinguish properly transformed cells versus non-transformed ones.

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PCR Analysis Confirmation assay to determine the presence of the t-PA gene inserted into tobacco transgenic plants was conducted through PCR amplification using two differentially forward and reverse primers. The forward primer was selected on the basis of the CaMV35S promoter (i.e., 5′CTTCAAAGCAAGTGGATTGATGTGATATCTCC3′), and the reverse one was designed according to the end of the t-PA gene (i.e., 5′TATCTCGAGCGGTCGCATGTTGTCACG3′). The following PCR conditions were employed: an initial cycle of 10 min at 94 °C followed by 35 cycles of 30 min at 94 °C, 1 min of annealing temperature at 59 °C, and 72 °C for 2 min, with a final extension step at 72 °C for 5 min. All PCR products were then separated by running on a 1.0 % agarose gel and visualized finally using ethidium bromide staining approach. Southern Blot Analysis The Southern blot technique was used to determine the copy number of the corresponding gene (i.e., t-PA) transformed into each transgenic plant. Total genomic DNA of transgenic T0 plants was first isolated according to Richardson et al. [30]. About 40 μg of genomic DNA was subsequently employed for digestion assay via HindIII, followed by separation on an agarose gel (0.9 %) electrophoresis system and transfer into a positively charged nylon membrane (Roche). Hybridization and detection were carried out by means of a DIG High Prime DNA Labeling and Detection Starter Kit (Roche) according to the manufacturer’s instruction. The middle region of the t-PA gene was used as probe (forward: 5′ TGGGGAACCACAACTACTGCAGAAAC3′ and reverse: 5′GAAACCTCTCCTGGAAGCAGTGGG3′) along with digoxigenin-dUTP as a detector-specific probe of t-PA. To produce the corresponding probe, PCR amplification was utilized then according to the following conditions: an initial cycle of 10 min at 94 °C followed by 35 cycles of 30 min at 94 °C, 1 min of annealing temperature at 59 °C, 72 °C for 2 min, and a final extension step at 72 °C for 5 min. RT-PCR Analysis Total RNA isolated from transgenic tobacco plant leaves (150 mg), using RNX™-plus solution according to the manufacturer’s instructions (http://www.cinnagen.com/Catalogue.pdf), was employed to survey the expression of the t-PA gene in positively transgenic plants. To verify the quantity and quality of the extracted RNA, all templates were first loaded on an agarose gel system (1.0 %) and simultaneously compared with 28S plus 18S-rRNA segments as indicators. Next, confirmed RNAs were treated using DNaseI (Fermentase, Lithonia) and subjected then to reverse transcription reaction using an M-MuLV Reverse Transcriptase (Fermentase, Lithonia) according to the manufacturer's instructions. In brief, the reaction mixture containing DNaseI was heated to 37 °C for 30 min, and then, the temperature was raised to 65 °C for 10 min to inactivate the enzyme. Afterward, cDNA was synthesized by the addition of reverse transcriptase enzyme to the mixture and heating it to 42 °C for 60 min and 70 °C for about 10 min. One pair of primers (forward: 5′TGGGGAACCACAACTACTGCAGAAAC3′; reverse: 5′ GAAACCTCTCCTGGAAGCAGTGGG3′) of the mid-t-PA cDNA amplifying 500 bp was used to confirm transcription in transgenic tobacco plants. PCR was performed in a thermocycler under an optimized condition as follows: 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min for a total of 30 cycles. The reverse transcription (RT)-PCR mixture was ultimately separated on a 1 % agarose gel and photographed.

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Enzyme-Linked Immunosorbent Assay In this part, an enzyme-linked immunosorbent assay (ELISA) was utilized to determine the proportion of t-PA in the total soluble proteins (TSP) in transgenic tobacco plants. Initially, a polystyrene microplate containing 96 wells (Costar, Cambridge, USA) was coated with 100 μg of total soluble proteins and incubated at 4 °C overnight. Each well was subsequently washed thrice, using phosphate-buffered saline (PBS). Next, blocking buffer (BSA) was added to each plate and incubated at 37 °C for 1 h. Similarly, each well was again washed thrice using washing buffer, and monoclonal antibody (raised in goat) was added to each well. After incubation at 37 °C for 1 h, the plate was again washed; PBS buffer was added and incubated for 1 h at 37 °C. Subsequent to the last washing step, 100 μL of tetramethylbenzidine was added to each well and incubated for 20 min under dark conditions. The enzyme reaction was stopped by quickly pipetting 100 μL of 1 M of HCl into each well. A microplate reader was used to read the absorbance at 450 nm. Notably, Alteplase (as commercial t-PA) was used as positive control. Bradford protein assay was used to measure protein concentration [33]. Zymography Zymography analysis, known as a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-like assay under a non-reducing condition, was performed for the assessment of the plasminogenolytic activity of the t-PA protein. The SDS-PAGE gels were copolymerized with plasminogen (Chromogenix, Milan, Italy) and gelatin as sequential substrates in order to detect proteolytic bands. Following electrophoresis at 4 °C for 5 h at a constant current of 8 mA, the gels were washed using 2.5 % Triton X-100 for 1 h by shaking at room temperature (25 °C) to remove SDS. Subsequent to gel incubation in 0.1 M glycine/NaOH (pH8.3) for 3 h at 37 °C, the gels were respectively stained and destained using Coomassie Brilliant Blue R-250 and destaining solution (acetic acid/methanol) overnight. The location of the peptide possessing enzymatic activity was revealed as a clear zone on a blue background followed by a white detectable band [34].

Results Molecular Analysis of Transgenic Tobacco Plants PCR The three chimeric constructs were transferred successfully into tobacco plants through A. tumefaciens transformation. In this context, the integration of the t-PA gene into the genomic DNA of tobacco plants was assessed first through PCR amplification using two separate forward and reverse primers designed on the basis of both CaMV35S promoter and t-PA gene which were exploited. In other words, regarding transformed tobacco plants using the pBI-SP-tPA construct, an expected amplified fragment of 1,948 bp (t-PA 1,698 bp; SP signal 120 bp and CaMV35S promoter amplified 130 bp) was detected successfully (Fig. 2a). With regard to tobacco plants that were transformed by the pBI-tPA-KDEL construct, a fragment with 1,828 bp (t-PA 1,698 bp; CaMV35S promoter 130 bp) was amplified (Fig. 2b). Finally, when genomic DNA of the tobacco plants transformed by the pBI-Ext-tPA construct was employed, an amplicon of 1,950 bp (t-PA 1,700 bp; extensin signal 120 bp; CaMV35S

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promoter 130 bp) was accurately amplified (Fig. 2b). These experiments were carefully fulfilled twice in order to substantiate the integration of the t-PA gene. As expected, there was no amplification in the wild-type genomic DNA under the same conditions (Fig. 2b). All these findings, consequently, demonstrate the successful production of transgenic plants in the experiment. Southern Blotting Overall, Southern blot analysis confirmed that a number of the current transgenic tobacco plants carry one or more copy numbers of the t-PA gene integrating successfully into the tobacco genomic DNA. In this regard, ten tobacco plants transformed via three different constructs were thoroughly investigated. Among the first five transgenic tobacco plants transformed by the pBI-tPA-KDEL construct, two samples had three and one copy number, respectively (Fig. 3, lanes 5 and 6). Furthermore, one out of three (lane 9) and also one out of two (lane 12) transgenic tobacco plants transformed by the pBI-Ext-tPA and pBI-SP-tPA constructs had just one copy number of the corresponding gene, respectively (Fig. 3). In addition, pBI-Ext-tPA plasmid was utilized as positive control subsequent to digestion via HindΙΙΙ (lane 7), whereas regarding negative control, a wild type of tobacco plant was employed with no amplification (Fig. 3). RT-PCR Following extraction of the total RNA of tobacco transgenic plants and RT-PCR amplification, the presence of the t-PA-specific transcription was accurately determined. The amplification products were observed to correspond to a specific major transcript of the expected size of the transgene (Fig. 4; lanes 3 to 14; lane 1, positive control). Despite the observation of expected amplification products of 500 bp for the t-PA gene in the actual transgenic plants, no bands were observed with the amplicons of non-transformed plants (wild type) grown as negative controls (Fig. 4; lane 2).

Fig. 2 a PCR products of tobacco transgenic plants using the pBI-SP-tPA construct (an expected band of 1,948 bp was observed). Left to right: lane 1, positive control; lane 2, wild-type control; lanes 3–10, tobacco transgenic plants; lane M, molecular marker. b PCR products of tobacco transgenic plants produced by both pBI-tPA-KDEL and pBI-Ext-tPA constructs (an expected band of 1,828 and 1,950 bp was detected, respectively). Left to right: lane 1, wild type; lane 2, positive control; lanes 3–8, tobacco transgenic plants using pBI-Ext-tPA construct; M, molecular size marker; lane 9, positive control; lanes 10–14, tobacco transgenic plants using the pBI-SP-tPA construct

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Fig. 3 Southern blot analysis of tobacco transgenic plants to determine the copy number of the t-PA gene transferring into each transgenic plant by pBI-SP-t-PA, pBI-t-PA-KDEL, and pBI-Ext-t-PA constructs using a digestion of genomic DNA of both wild-type and tobacco transgenic plants with HindΙΙΙ. a Before and b after blotting assay. Left to right: lane 1, wild-type plant; lanes 2–6, transgenic plants using the pBI-t-PA-KDEL construct; M, molecular marker; lane 7, positive control (plasmid); lane 8, negative control (water); lanes 9– 11, tobacco transgenic plants transformed with the pBI-Ext-tPA construct; lanes 12 and 13, tobacco transgenic plants transformed with the construct pBI-SP-t-PA

ELISA Assay ELISA assay, as another screening scenario, was employed to find positively transgenic tobacco plants in one hand and to make probable differentiations in relation to the expression level (i.e., immunoactivity) of rt-PA among a total of 30 transgenic tobacco plants generated with three distinct constructs including pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA. The method, as a result, demonstrated that the recombinant protein extracted from all 30 transformants exhibited normal immunoactivity. Nevertheless, as shown in Fig. 5, the expression level of rt-PA followed a fluctuating trend: the highest expression level of rt-PA was exhibited by the T8 transgenic plant line generated from pBI-tPA-KDEL, while T28, produced by pBI-SP-tPA, had the lowest amount of rt-PA production (Fig. 3a). In addition, regarding SP, KDEL, and extensin signals, the mean values of t-PA expression were calculated as 0.50, 0.68, and 0.69 μg/mg of the TSP, respectively. Unsurprisingly, when non-transformed plants (wild type) were used as negative control, the amount of rt-PA production led to the minimum level that is zero. Zymography Assay To determine the molecular weight of rt-PA and measure the activity of the plasminogen activator in the leaf extracts of tobacco transgenic plants, zymography assay was utilized as

Fig. 4 RT-PCR products amplified from the total RNA of transgenic plants. Primers designed for PCR amplification of the t-PA gene specifically amplified DNA fragments of 500 bp. Left to right: lane 1, positive control containing the plasmid; lane 2, wild-type plant (as negative control); lanes 3–6, 7–10, and 11–14, transformed tobacco plants using pBI-SP-t-PA, pBI-Ext-t-PA, and pBI-t-PA-KDEL constructs, respectively; M, molecular size marker

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Fig. 5 Graphical pattern of ELISA assay. Ten tobacco plants transformed successfully via three different constructs including pBI-SP-t-PA (lanes 1–10), pBI-t-PA-KDEL (lanes 11–20), and pBI-Ext-t-PA (lanes 21– 30) were used followed by wild-type tobacco plant as negative control (lane 31)

the last part of the current investigation. According to our results, interestingly, when the transgenic plants generated from the construct namely pBI-tPA-KDEL (lanes 1and 2), pBISP-tPA (lanes 3 and 4) and pBI-Ext-tPA (lanes 5–7) were utilized followed by a clear zone pattern (Fig. 6). Further, the activity validation of rt-PA was also confirmed as long as Alteplase (as commercial t-PA) was used as positive control (lane 11; an approximate size band of 63 kDa), whereas the desired cleared zones were not obtained in control reactions (lanes 8–10) where the template was the leaf extracts from the wild type of tobacco plants. The sharp clear zones on the blue background of the zymography gel, overall, indicated the real plasminogenolytic activity of the rt-PA protein.

Discussion The current biofarming-based investigation was designed to produce three different chimeric gene constructs derived from t-PA (i.e., pBI-Ext-tPA, pBI-SP-tPA, and pBI-tPA-KDEL), subsequently introduce them into tobacco plants through an Agrobacterium-mediated transformation system, and finally survey expression levels of rt-PA in all transformants by means of several molecular- and biochemical-based approaches. In this context, following PCR amplification to validate the accurate integration of the t-PA gene into the tobacco

Fig. 6 Gelatin-based zymography pattern to appraise the plasminogenolytic activity of the rt-PA protein. Left to right: lanes 1 and 2, tobacco plants transformed by pBI-tPA-KDEL; lanes 3 and 4, tobacco plants transformed by pBI-SP-t-PA; lanes 5–7, tobacco plants transformed by pBI-Ext-tPA; lanes 8–10, tobacco wild-type plant; lane 11, positive control with Alteplase (a size band of 63 kDa was observed successfully)

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nuclear genome, RT-PCR was also utilized and could successfully verify the presence of the t-PA-specific transcription. The assay of Southern blot analysis, in addition, could verify the accurate integration of rt-PA into the tobacco transgenic plants, even though the positively transgenic plants carried different copy numbers of the t-PA gene, ranging from one to three (Fig. 3). It is noticeable that the higher number of copies (i.e., more than one copy) of a certain gene may be associated with a series of unintended consequences such as silencing due to methylation process. Hence, as complementary assays, two popular biochemical assays called ELISA and zymography were employed, both of which, overall, confirmed the accurate expression and enzymatic function of the rt-PA, respectively. With respect to ELISA assay, as described earlier, the mean values of rt-PA expression (produced by SP, KDEL, and extensin signals) were calculated as 0.50, 0.68, and 0.69 μg/mg of the TSP, respectively. The following points could be accordingly concluded: (1) despite the rate of expression level, rt-PA was integrated and surprisingly expressed successfully in all three cell spaces of the transgenic tobacco plants, depending on the corresponding signals; (2) there were no significant differences between the average values of t-PA expression of transgenic tobacco plants produced by the pBIExt-tPA construct and those derived from the pBI-tPA-KDEL construct, both of which, however, were considerably superior than that of the pBI-SP-tPA construct. Such contradictious results, presumably, could be due to different conditions in which they are expressed. For instance, following targeting recombinant proteins via both extensin and KDEL signals, they can be precisely transferred into an apoplastic space or ER where they are protected from degradation by cytoplasmic proteases. The yield of the desired product, as a result, may be further enhanced; (3) among individuals produced only by each construct, as shown in Fig. 5, a fluctuating trend could be observed, probably arising from probable differences in positional insertion of the ectopic gene. Anyway, consistent with the previous investigations, depending on either the capability of used promoters for expression of specific genes or plants to be utilized for transformation, the value of recombinant antigens in positively transgenic plants has been usually recorded as 0.002 % to 7 % of the total soluble protein [35–38]. Regarding the expression level of rt-PA in tobacco plants, however, a lower level (a range between 0.0002 % and 0.0014 % of the total soluble protein) has been reported by Hahn et al. [15]. More recently, using hairy root system, a significant improvement to produce rtPA (33-fold higher than that of Hahn et al. [15]) was reported by Kim et al., [29], ranging from 0.05 % to 0.08 % of the total soluble protein. In the current study, surprisingly, a slight enhancement was detected varying from 0.02 % to 0.115 %. Even though the rate of recombinant t-PA produced in this study was significantly higher than that of previous investigations [15, 29, 35], such production values, nevertheless, seem to be minor probably due to the pivotal role of proteolysis on the overall efficiency of the plant system [15, 29, 35], still needing to be intensified (see above). The enzymatic activity of a given protein (here, rt-PA) could be measured via an assay called gelatin zymography method [19, 27, 28, 35]. The method is commonly designed on the basis of the in vivo activity of t-PA, converting plasminogen to plasmin as a result [39, 40]. Interestingly, depending on the kind of construct, clear zones with three different molecular masses (see the “Results” section) were observed similar to commercial t-PA (Alteplase). These results, which were in agreement with previous studies [19, 27, 28], imply the fact that the current rt-PA has enough potential to express and function as much as commercial t-PA (Alteplase), and could be accordingly extracted and purified as an alternative and cost-effective source on the account of generating a large-scale production of t-PA commercially.

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Conclusion To sum up, in this study, tobacco transgenic plants were developed successfully using three different signals in order to produce a human tissue-type plasminogen activator as a biologically active and medically important enzyme. Our current results, overall, exhibited that the tobacco plant, due to its several unique characteristics, can be assigned, alternatively, as a reliable and cost-effective bioreactor in the large-scale production of industrial enzymes and pharmaceutical proteins including t-PA. Meanwhile, despite the successful expression and accurate function of rt-PA even in different cell spaces, to produce rt-PA in a large quantity, additional efforts followed by developing novel tools need to be taken into account.

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