Plasmids: recombinant pET15b/reteplase was previously prepared at the School of Pharmacy of Isfahan University of Medical Sciences ⁽⁸⁾. The pBAD/gIII plasmid and bacterial strains E. coli Top10 were obtained from Pasteur Institute, Iran.

Luria-Bertani (LB) media was prepared according to the guidelines in the laboratory manual offered by Sambrook and Russell ⁽⁵⁾. Screening based on antibiotic resistance was performed on LB agar plates containing ampicillin (100 µg ml^-1) which was obtained from Sigma, Germany ⁽¹²⁾.

One hundred µl of CaCl₂-competent E. coli DH5α (Cinnagen, Iran) cells were transformed with recombinant pET15b/reteplase plasmid. The transformed cells were spread on Luria-Bertani agar plates containing 100 mg of ampicillin (Sigma, Germany) per ml. After cultivation at 37°C for 24 hr, some colonies were selected for plasmid minipreparations using kit method (purchased from Fermentas Co., Poland). Plasmids were digested with NcoI and BamHI restriction enzymes to obtain reteplase cDNA inserts. On the other hand, pBAD vector was digested with NcoI and BglII enzymes. Vector and insert (molar ratio of 3:1, vector to insert) were ligated with T4 DNA ligase. Subsequently, E. coli Top10 bacteria were transformed using heat shock method (39°C, 1 min) and were spread on LB agar plates containing 100 µg/ ml ampicillin and then incubated overnight at 37°C. Afterwards, the obtained recombinant pBAD/gIII plasmids were sequenced (Biotechnology Kosar using the Analyzer Genetic Device and Capillary Base).

A single clone of E. coli Top10 cells containing recombinant plasmid was cultured in 5 ml Luria Bertani medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) at 37°C, 180× g overnight. Then, 1 ml of this culture was inoculated into 100 ml of Luria-Bertani medium supplemented with ampicillin (100 µM) until OD600 of 0.4-0.6 was reached. Various concentrations of L-Arabinose was then added (0.2, 0.02, 0.002 and 0.0002%). Cells were incubated at 37°C, 180× g for an additional 4 hr and harvested by centrifugation at 5,000 g at 4°C for 10 min and the final product was stored at -20°C ^{(13, 14)}.

Periplasmic proteins are proteins secreted into the periplasmic space located between the outer and inner membranes of E. coli. Proper secretion is possible only when the protein of interest has an N-terminal signal peptide that is cleaved following translocation. Cultured E. coli cells containing recombinant plasmid were centrifuged and the obtained pellet was resuspended in 30 mM Tris, 20% sucrose, pH=8.0, at 80 ml/gm wet weight. After incubation on ice for 10 min, 500 mM EDTA was added dropwise to the final concentration of 1 mM. The cells were placed on ice for another 5-10 min with gentle agitation. These cells were centrifuged at 8000 ×g for 20 min at 4°C and the supernatant was discarded. The pellet was resuspended in ice-cold 5 mM MgSO₄, stirred for 10 min in ice bath and centrifuged at 8000 ×g for 20 min at 4°C. The supernatant (sample A) contained the periplasmic proteins and the remaining sediment (sample B) was stored at 4°C ^{(5, 15)}.

For this purpose, about 25 gr of the cells were resuspended in 580 ml of 0.1 mol/L Tris and 20 mmol/L EDTA and homogenized with a shearing rod. Lysozyme at 0.25 mg/ml was added followed by incubation for 30 min on ice. Thereafter, centrifugation was carried out for 50 min at 4°C (15000 ×g). The pellets were resuspended in 300 ml of 0.1 mol/L Tris, 20 mmol/L EDTA and 2.5% V/V Triton X-100 and homogenized. After centrifugation, the pellets were resuspended in 300 ml. 0.1 mol/L Tris, 20 mol/L EDTA and 0/5% v/v Triton X-100 and homogenized. The samples were then centrifuged for 30 min at 4°C and 15000×g and the pellets were resuspended in 250 µL of 0.1 mol/L of Tris and 20 mmol/L EDTA ^{(16, 17)}. The prepared inclusion bodies were stored at -20°C.

Proteins were sequentially extracted from the prepared inclusion bodies by resuspension in 10 ml of Tris 25 mM, EDTA 10 mM, containing 1% Triton X-100 and guanidine HCl 6-8 M ⁽¹⁸⁾. The reducing agent and buffer components were separated by dialysis against 6 mol/L guanidine hydrochloride (pH=7) at 4°C ^{(4, 19)}.

The solubilized samples were incubated in Tris 0.1 mol/L (pH=8.5); guanidine hydrochloride 6 mol/L; EDTA 2 mmol/L and DTT 0.3 mol/L for 3 hr at ambient temperature. We tested 3, 6, 12 and 24 hr retention times and our results showed that there was no difference between these times and therefore we used 3 hr for retention time. Subsequently, the pH of the solution was adjusted to 7 with concentrated hydrochloric acid. Refolding of the protein took place by dilution with 0.1 mol/L Tris (pH=10.5), 0.5 mol/L L-argenine, 1 mmol/L EDTA, 6-8 mol/L guanidine hydrochloride and 1 mg/ml bovine serum albumin. Then, the samples were incubated for 24 hr at 20°C. The reducing agent and buffer components were separated by dialysis against 6 mol/L guanidine hydrochloride (pH=7) at 4°C ^{(4, 19)}.

Harvested cells were washed with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.05% Tween 20, pH=7.4). Next, the samples were boiled for 5 min at 70°C and electrophoreses on a 12% (vol/vol) SDS-PAGE analysis. Subsequently, the gels were stained with Coomassie brilliant G250.

In this project, the Q-sepharose Fast Flow was used for separation and purification of reteplase. Q-sepharose Fast Flow was equilibrated with Tris buffer (pH=9) and the protein samples were added to the column and eluted in a step wise manner with equilibration buffer containing 0.5 M to 1 M NaCl. This elution step was performed until no further proteins were eluted from the column. Then, the samples were analyzed using SDS-PAGE ^{(12, 20)}.

After SDS-PAGE, the proteins on the gel were transferred onto a nitrocellulose membrane. After incubating the blot with blocking buffer (5% non-fat dry milk in TBS) overnight at 4°C, tissue plasminogen activator antibody (anti t-PA, 1:250 to 1:1000 in blocking buffer) was added and incubated for 1 hr at room temperature. Subsequently, three washes with the blocking buffer were performed and anti-rabbit IgG-HRP conjugate (Roche, Germany) secondary antibody (diluted 1:5000) was added (incubated at room temperature for 1 hr). The membrane was washed three times with 20 ml TBS-Tween for 10 min. The TBS-Tween solution was then removed, the ECL solution was added (Amersham, USA) and the bands were detected on a common X-ray film.

Activation of plasminogen by reteplase was detected by Chromogenic Activity Assay Kit. Briefly, assay diluention (50 µL), plasminogen (10 µL), plasmin substrate (20 µL) and 20 µL of tPA standards or the samples were added to the supplied 96-well plate. The plate was incubated at 37°C in a humid incubator and the absorbance at 405 nm for each sample was measured.

Recombinant pET15b plasmid containing reteplase sequence was digested with NcoI and Bam HI restriction enzymes while pBAD/ gIIA plasmid was digested with NcoI and BglII. Molecular weight of pBAD/gIIA is 4145 nucleotides and reteplase contains 1128 nucleotides (Figure 1). After ligation and transformation, the presence and correct orientation of the insert was evaluated using NcoI and HindIII restriction enzymes. As shown in Figure 1, the recombinant plasmids contained the correctly oriented insert. Sequencing of this plasmid also confirmed the presence of reteplase cDNA in the pBAD/ gIIA plasmid.

After the transformation of E. coli Top10 bacteria with pBAD/gIIA plasmid containing reteplase gene, the effect of different concentrations of L-Arabinose (0.2, 0.02, 0.002 and 0.0002%) on reteplase expression was evaluated (1 hr, 2 hr, 3 hr and 4 hr at 25°C, 30°C and 37°C incubation times and temperatures, respectively). The expressed proteins were electrophoresed using SDS-PAGE (Figure 2). Maximum production of reteplase occurred under the following condition: 0.0002% L-Arabinoze (as inducer), incubated at 37°C for 2 hr.

Protein production was induced by L-Ara-binose (0.0002% for 4 hr). Subsequently, proteins were isolated from the periplasmic space (samples A and B, supernatant and pellet, respectively) and were analyzed using SDS-PAGE. A protein with an estimated size of ∼39 kDa was observed in samples A and B (Figure 3).

Regarding isolation of inclusion bodies, after inducing with L-Arabinose 0.0002% for 4 hr, extraction of inclusion body was performed and the obtained pellets were analyzed by SDS-PAGE. As shown in Figure 3, a strong 39 kDa band was observed.

The obtained inclusion bodies were solubilized and refolded for 24 hr. After dialysis, purification of reteplase was performed using Q-sepharose fast flow column. This column was equilibrated with Tris buffer (pH=9) and proteins were eluted in a step wise manner with equilibration buffer containing 0.5 M to 1 M NaCl (speed of about 1 ml/min). A strong band was observed in fractions obtained after addition of 0.6 M NaCl (Figure 4).

After purification, the obtained reteplase was confirmed by Western blotting using anti-His antibodies (Figure 5).

The activity of the obtained enzyme was analyzed using standard t-PA enzyme activity kit (Table 1). The change in the absorbance of pNA (para nitroaniline) in the reaction solution at 405 nm was directly proportional to the t-PA enzymatic activity. The concentration of tPA standard was 8000 units that is equal to 40 mg/ml. The concentration of samples was measured using standard curve of BSA (albumin serum) and it turned out to be 12 µg/µL.