Recursive PCR and Primer Design
For synthesis of optimized IGF-1 (IGF-1s) gene, four primers were designed: two external primers of 56 bp and two internal primers of 100 bp. All the primers have an overlapping region of 20 bp. The 5' external primer was engineered to include the sequence of the TEV enzymatic cleave site and the 3' primer contained the NotI restriction site. In recursive PCR reaction, the external oligonucleotides were in higher concentration than the internal (20–30 pmol of the external primers and 0.2–0.3 pmol of the internal primers). The lower concentration of the internals oligonucleotides assisted in avoiding unwanted products.
Two different parts were used in the recursive PCR [49, 50]. In the first part, the reaction were run through 10 cycles using the following temperature sequence: 94°C for 30 seconds to denature the DNA, 55°C for 30 seconds for annealing primes, and 72°C for 1 minute to synthesize DNA. An incubation period of 7 minutes at 72°C followed after the cycles ended. The primers were designed to have an annealing temperature of 55°C to avoid unspecific binding of the primers. The second part consisted of 30 cycles, denaturing the DNA for 30 seconds at 94°C, then primers annealing for 30 seconds at 65°C, followed by DNA synthesis for 7 minutes at 72°C. The PCR product was run on 1.5% agarose gel at 65 volts for 55 minutes to visualize amplified products. The IGF-1s was cloned into the pBluescript KS II, and E. coli cells were transformed with this vector.
Bombardment and Selection of Transgenic Lines
Sterile leaves were bombarded using the Bio-Rad PDS-1000/He biolistic device [51, 52]. The bombarded leaves were incubated in the dark for 48 hours and then cut and placed in RMOP medium with 500 μg/ml of spectinomycin.
The plant DNA was extracted from leaves using the Qiagen Dneasy Plant Mini Kit (Quiagen). The 3P and 3M primers were used to perform PCR on transformed and untransformed plants [51–53]. Samples were run for 30 cycle with the following sequence: 94°C for 1 min., 65°C for 1.5 min., and 72°C for 2 min. PCR products were analyzed on 0.8% agarose gel.
Southern Blot Analysis
The plant DNA of the transgenic and wild type tobacco plants were digested with BglII, and separated on 0.8% agarose gel and transferred to a nylon membrane. The 0.8 kb probe was generated by digesting pLD-CtV2 (that contains the trnI and trnA genes) vector with BamHI and BglII and was labeled with 32P (Amersham). The probe was hybridized with the membrane using the QUICK-HYB hybridization solution and protocol (Stratagene).
ELISA was used to quantify the IGF-1 expression levels in different transgenic lines. Different concentrations of 100 mg leaves (transformed and untransformed plants) were ground with liquid nitrogen. Bicarbonate buffer, 500 μl, pH 9.6 (15 mM Na2CO3, 35 mM NaHCO3, and 0.1% Tween 20, pH 9.6) was used to resuspend the ground mixture and incubated overnight at 4°C. Diluted sample (1:3000) was added in each well (100 μl) of the plate and this was done in duplicate. Bicarbonate buffer was used as blank. The plate was incubated overnight at 4°C. After washing the wells thrice with washing buffer, PBST (PBS and 0.05% Tween 20), mouse anti human IGF-1 diluted 1 μg/ml in 0.01 M PBST containing 0.3% milk (100 μl/well) was added and incubated for 2 h at 37°C. The wells were washed and incubated with 1:10,000 goat anti mouse IgG-alkaline phosphatase conjugate in 0.01 M PBST containing 0.3% milk (100 μl/well) for 2 h at 37°C. The plate was developed with TMB substrate (100 μl/well, American Qualex) for 30 minutes at room temperature and the reaction was stopped by addition of 50 μl/well of 2 M sulfuric acid and the plates were read at 405 nm. For a standard curve, purified commercially available human IGF-1 (R&D Systems) was diluted with bicarbonate buffer to concentrations between 3 and 25 ng/ml and processed as above. Total soluble plant protein concentration was determined using the DC Protein Microassay (Bio-Rad). IGF-1 expression levels were calculated as a percentage of the total soluble protein.
One hundred mg of tobacco leaves were ground in liquid nitrogen and resuspended in 200 μl of extraction buffer (200 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 4 mM PMSF) . Leaf extracts were boiled for 5 minutes in the sample buffer (0.5 M Tris-HCl, pH 6.8, 2.5 ml glycerol, 10% SDS, 0.5% bromophenol blue reached a total volume 9.5 ml with water) (Bio-Rad). All samples were electrophoresed in 15% resolving and 4% stacking gels using the buffer system of Laemmli. The membrane was blocked for 20 minutes at room temperature with PBS and 3% non-fat milk (PBS-milk). Then, the blot was incubated with anti-IGF-1 (Upstate Biotechnology, diluted in PBS-milk until it achieved a final concentration 1 μg per ml) overnight at 4°C. The membrane was washed twice with water. The secondary antibody used was a Goat Anti Mouse IgG conjugated to Horseradish Peroxidase (American Qualex Antibodies) at a 1:5000 dilution, and was added to membranes in blocking solution and incubated for one hour. The blot was washed with water. A final wash was done for 5 minutes in PBS with 0.05% Tween 20. Development was performed by the chemiluminescent method (Pierce).
Following elution from the immuno-affinity column, the protein samples were concentrated by speed vac evaporation and diluted 1:2 in 1× Gel-loading buffer (50 mM Tris, 2% SDS, 0.05% Brome-Phenol Blue, 10 v/v% glycerol and 2 μl DTT (dithiothreitol). The samples were heated at 95°C for 5 min, and 20 μl (protein conc. 500 μg/ml) of the sample was loaded onto the gel. Electrophoresis was performed at 160 V-, in 1× Laemmli running buffer (5× Laemmli: 500 ml-in water solution 7.5 g Tris, 47 g glycine, 2.5 g SDS). Capillary blotting of proteins was carried out overnight in transfer buffer (3 g Tris, 14.4 g glycine, 200 ml methanol per liter). The nitrocellulose membrane was washed for 5–10 min with TST-buffer (4.5 g NaCl, 250 μl Tween 20, 25 ml in 500 ml 1 M Tris, pH 7.5), then blocked in TST containing 5% dry milk (TST = 5 g dry milk in 100 ml) for 30 min in glass tubes of the DNA hybridization equipment, with slow rotation. The primary antibody (goat a-human IGF-I, SIGMA Cat#8773) was diluted1:1250 in TST (with 2.5% dry milk); membranes were rinsed in distilled water. The primary antibody (10 ml) was added to membranes, and incubated for 2 h at room temp with gentle shaking. Membranes were washed 5 times in distilled water followed by 3 × 5 times in TST, (50 ml). The secondary antibody (Rabbit a-goat alkaline phosphatase-conjugate, Sigma Cat#4049), or monoclonal a-goat alkaline phosphatase-conjugate- Sigma Cat#8062) was diluted in TST buffer (1:10000, 20000, 50000), added to blots and incubated with gentle rotation on an orbital shaker for 1 h. Blots were washed 3 × 5 times in TST, followed by 5 times in distilled water. Immunoblots were developed for 20–40 min in NBT substrate: made up of 5 mg NBT (nitrotetrazolium blue, Sigma) diluted in100 μl of 70% DMF (dimethyl-formamide) solution; 82 μl BCIP (5-bromo-4-chloro-3-indolyl phosphate disodium salt) was added to 10 ml substrate buffer (100 ml: 10 ml 1 M Tris, 2 ml 5 M NaCl and 5 ml 1 M MgCl2). Blots were stained at 4°C overnight. The reaction was stopped by dilution of the substrate in cold tap water.
Purification of IGF-1 from transplastomic leaves
Transformed tobacco leaves (50 g) were ground to a fine powder in a liquid nitrogen in pre-cooled mortar. The plant powder was thawed by addition of two volumes (100 ml) of cold Extraction Buffer A (50 mM Tris HCl, pH 8.0, containing 200 mM NaCl, 100 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1% Nonidet P-40). The homogenate was centrifuged in 50 ml Oakridge tubes at 9,000 rpm, 10 min, at 4°C in a pre-cooled SA-600 rotor, in a Sorvall RC-5B centrifuge to remove cell debris. The non-IGF-1 plant proteins in the supernatant were precipitated by the addition of solid ammonium sulfate to 30% saturation (w/v), while the IGF-1 remained in the soluble fraction. The mixture was centrifuged at 9,500 rpm in a Sorvall SA-600 rotor at 4°C for 30 min. The supernatant containing the IGF-1 was raised to 65% saturation with ammonium sulfate and the mixture was centrifuged as before. The IGF-1 was now precipitated in the pellet. The pellet was resuspended in a small volume (3–4 ml) of Extraction Buffer B (25 mM Tris-HCl, pH 7.5, 0.01% thiodiglycol and 10 μM PMSF) – 10×. The mixture was dialysed against 1.0 liter of extraction buffer B at 4°C for 6 hrs in dialysis tubing with a molecular weight cutoff of 7,000 kDa or less. The dialysate was centrifuged as before at 9,500 rpm in the SA-600 rotor for 15 min to sediment insoluble proteins. The supernatant containing the IGF-1 was adjusted to 0.15 M NaCl and loaded onto a IgG-Fast Flow 6 Sepharose (Sigma) immunoadsorbent column (10 ml bed vol), equilibrated with TST buffer (50 mM Tris-HCl, pH 7.6, 120 mM NaCl, 0.05% Tween 20). Proteins other than ZZ-IGF were eluted from the column by washing with 10–20 column volumes of TST buffer. The Tween 20 detergent was removed from the column by washing with 2 volumes of 5 mM ammonium acetate, pH 4.8. The ZZ-IGF-1 fusion protein was eluted from the column with 0.4 M acetic acid, pH 3.4 at a low linear flow rate (20 cm/hr) to obtain a sharp IGF-1 peak. The column eluate was collected (2.0 ml fractions), and monitored (OD280) for the appearance of ZZ-IGF-1 protein which eluted after the column volume. The fractions containing the ZZ-IGF-1 protein were pooled and the protein concentration was determined spectrophotometrically by comparison of the OD260/280 nm ratio, or more precisely by Bradford protein assay. Residual salt was removed by dialysis for 2–3 hrs against 1 l of 5 mM ammonium acetate, pH 6.0, at 4°C using dialysis tubing with a 3.5 kDa cutoff (Pierce). A sample of the ZZ-IGF-1 protein was subjected to electrophoresis on a 15% acrylamide gel to examine the molecular weight of the ZZ tagged IGF-1.
The ZZ-linker was cleaved from the IGF-1, by dissolving 2.0 mg of previously isolated tobacco ZZ-tagged IGF-1 fusion protein in 1.0 ml of ZZ Buffer without hydroxylamine (0.2 M Tris-HCl, pH 9.2, 1 mM EDTA). Cleavage of the ZZ moiety from the IGF-1 was carried out by addition of an equal volume of ZZ buffer (0.2 M Tris-HCl, pH 9.2, 2 M hydroxylamine, 1 mM EDTA).containing 2.0 M hydroxylamine. The mixture was incubated at 45°C for six hours and the reaction was terminated by lowering the pH to 6.0 with acetic acid. Cleaved IGF-1 was estimated by separation of the two forms by acrylamide gel electrophoresis. Hydroxylamine was removed from the mixture by first lowering the pH to 6.0 with glacial acetic acid (measured with pH paper), followed by dialysis of the mixture for 2 hrs at 4°C in 1.0 liter of 50 mM ammonium acetate buffer, pH 6.0. The ZZ-tagged-IGF-1/IGF-1 mixture was lyophilized yielding a tan powder. After resuspension of the powder in a small volume (0.5 ml) of distilled water, the mixture was separated by polyacrylamide gel electrophoresis on a 15% acrylamide gel in a BioRad minigel electrophoresis apparatus for 1 hr, at 100 Volts. The protein bands (2) representing IGF-1 and ZZ-tagged-IGF-1, were stained by immersion of the gel in "gelcode" (Pierce), bromophenol blue stain for 2 hr. The gel was destained in distilled water prior to photographic documentation.
To remove the contaminating ZZ ligand and uncleaved ZZ-IGF-1 from the IGF-1 molecules, the cleavage mixture was passed over a small (3.0 ml) IgG-sepharose column previously washed with 10× vol of TST, 2× vol of TS and 2× vol of 5 mM ammonium acetate, pH 4.5. IGF-1 molecules passed through the column following the void volume (1.0 ml, determined by the elution of blue dextran), while the free ZZ domains and uncleaved ZZ-IGF bound to the IgG -sepharose. After collection of the column eluate (1.0 ml fractions), the amount of IGF-1 protein was measured by Bradford protein assay and the protein diluted with sterile water, divided into aliquots and frozen at -20°C for use in biological assays or structural studies.
Cell Proliferation Assays
The biological activity of partially purified human IGF-1 preparations isolated from tobacco chloroplasts was established by measurement of IGF-1 effects on mammalian cell proliferation. Briefly, human megakaryoblastic (HU-3) cells carrying the IGF-1 receptor were cultured in RPMI-1640 medium + 10% calf serum. Cells in the logarithmic phase of growth were seeded into 96-well plates at a concentration of 25,000 cells/well in 50 μL RPMI-1640 containing 0.1 BSA and 0.5–1% calf serum. At the beginning of the assay, commercial human IGF-1 (Sigma), or IGF-1 partially purified from tobacco chloroplasts (50 μL) was added to each well at concentrations from 0.001 μg/ml to 10 μg/ml. The cell cultures were incubated at 37°C for 48 hours and a portion of each culture was stained with Trypan Blue dye to quantify cell viability prior to measurements of cell proliferation as determined most reliably by cell counting by hemocytometer.
Murine 3T3 fibroblasts also carrying the IGF-1 receptor were maintained in DMEM medium and seeded in 96-well plates from 2,000 cells/well to concentrations as high as 20,000 cells/well in 50 μL DMEM + 0.1% BSA containing 0.1–1% calf serum. IGF-1 (50 μL) was added in ascending concentrations from 0.001 to 10 μg/ml. Forty eight hours later (some time after 7 days) cell proliferation was measured by the CyQUANT NF Cell proliferation assay (Invitrogen Inc., San Diego, CA) and by hemocytometer counting. Proliferation was measured by fluorescence determination of DNA replication based on intercalation of a fluorescent dye into double stranded DNA. Cell proliferation was determined according to the instructions supplied by the manufacturer. Red, blue and green lines represent the best fit of the respective data points for each proliferation assay treatment. The commercial human IGF-1 used in the assay was Sigma-Aldrich Cat#3769 (10 μg-0.001 μg/ml).