Introduction(bits and pieces):
The general utilization of transgenic plants or refined cells as bioreactors for financial generation of pharmaceuticals holds incredible guarantee and a few plant-based frameworks have been produced for delivering root exudates, along with soluble recombinant proteins , phyllosecretion,seed oil-bodies, suspension refined cells and completely genetically modified plants. These frameworks have their own particular positive points and weaknesses regarding expense, timescale, creation and capacity. (1)
Lysosomal storage diseases (LSDs), that is accountable for more than 50 issues, are agreeable to enzyme replacement treatments. Then again, the present techniques used to economically deliver recombinant lysosomal enzymes for this reason, most usually Chinese Hamster Ovary cells and human fibroblasts which are restrictively expensive. Plant bioreactors holds considerable guarantee for financial generation of practical human a-L-iduronidase (hIDUA; glycosaminoglycan a-L-iduronohydrolase), the enzyme present inadequately in the human LSD, Mucopolysaccharidosis I. Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease (LSD) in humans created by an inadequacy of the lysosomal enzyme a-L-iduronidase; in seriously affected humans this hereditary sickness prompts demise in infancy as a result of significant skeletal, cardiovascular and neurological unsettling influences that occurs from a recurring inefficiencey to successively degrade the glycosaminoglycans, heparan sulfate and dermatan sulfate. Aldurazyme1, recombinant human a-L-iduronidase derived from a culture of Chinese Hamster Ovary (CHO) cells, has been utilized as a part of enzyme replacement therapy for treating MPS I. Then again, the expense of CHO cell-culture derived enzyme (Aldurazyme1) is very high for general application that requires 260,000 USD per patient each year, or additionally relying upon the weight of the individual and the particular dosage. Therefore, plant inferred hIDUA may be an option for monetary creation of the enzyme to be utilized for restoration therapy.
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In this study, we have tried and utilized genetically modified tobacco BY-2 cells as bioreactors for the creation of active human α-Liduronidase (hIDUA). The expression of human α-lactalbumin, a noteworthy milk protein, in genetically modified tobacco plants is effective with the utilization of the local (i.e. human) signal peptide; although in this study, we found that a plant signal peptide prompted elevated amounts of hIDUA protein in the cultured media of transgenic tobacco BY-2 cells. The yield and working of the discharged 78 kDa glycosylated hIDUA from long-term cultures of transgenic BY-2 cell lines were as high as 10 mg/mL media and 53,000 pmol/min/mg proteins. Also, the expense of culture and ensuing accumulation of 1 L medium (equal to pretty nearly 10 mg aggregated IDUA) from BY-2 cell lines expressing IDUA is assessed to be under 10 USD. In this manner, this transgenic BY-2 cell line shows an appealing stage for monetary creation and simple downstream decontamination of hIDUA.
The entire methodology for the production of α -iduronidase is explained in the following steps:
- Antibody generation
Human IDUA synthetic peptides CSASGHFTDFEDKQQVFE (peptide 1) and CSPDGEWRRLGRPVFPTAE (peptide 2) were joined with keyhole limpet haemocyanin (KLH) and used as antigens to inject rabbits for antibody generation at the animal house of the Chinese University of Hong Kong. The generated antibodies were furthermore affinity-purified with a CNBr activated Sepharose column affixed with synthetic peptides or purified recombinant hIDUA proteins. Affinity-purified hIDUA antibodies were used at 4 µg/mL in various applications.
- Recombinant DNA constructs
For the construction of SPp-IDUA and Spi-IDUA, the sequences which encoded the mature hIDUA protein plus sequences which encoded the signal peptide of proaleurain or encoded the local human IDUA signal peptide, respectively, were subcloned into the binary vector pBI121. The resulting construct SPp-IDUA or Spi- IDUA thus consists of the CaMV 35S promoter, followed by the proaleurain or the human IDUA signal peptide sequences, the IDUA (mature protein) sequences, and the NOS terminator and 30 flanking region. The constructs were analyzed by both sequencing and restriction mapping.
- Transformation of BY-2 cells
For Agrobacterium-mediated transformation, plasmids SPi-IDUA and SPp-IDUA were firstly introduced into Agrobacterium with the help of electroporation before the transfection of wild type tobacco BY-2 cells. BY-2 cells were maintained in MS liquid medium at room temperature in a shaker set at 125 rpm. Transfected BY-2 cells were shifted on MS medium (Sigma–Aldrich) consisting of kanamycin (50 µg/mL) and cefotaxime (250 µg/mL) and incubated at room temperature for 3–4 weeks until transformed colonies were easily seen. Transgenic cell lines with antibiotic resistance (5–10 per construct) were further shifted into MS liquid medium having kanamycin to start suspension cultures and used for following analysis. Transgenic BY-2 cell lines were maintained in both liquid and solid culture via subculturing.
- Precipitation of secreted IDUA and western blot analysis
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For precipitation of the protein from the cultured media of transgenic BY-2 cells, culture medium was mixed with four parts methanol, one part chloroform and three parts water, which was followed by centrifugation at 14,000 rpm for 10 min. The resulting precipitates were again suspended in four parts methanol and centrifuged again. The protein pellet was then again suspended in SDS loading buffer for separation of protein via SDS-PAGE after which western blot analysis was done using the freshly produced IDUA antibodies at 4 µg/mL.
- Endoglycosidase digestion of secreted IDUA
Typical Endoglycosidase H digestion of precipitated IDUA proteins from the cultured media of genetically modified BY-2 cells was carried out. The pellet of protein was dissolved in water before the mixing with 10 glycoprotein denaturing buffer to a final measurement of 10 mL. Subsequent denaturation was carried out by heating the reaction for 10 min at 100 ºC. 2 µL of 10x G5 reaction buffer, 4 µL H2O and 4 µL Endo H were then added to reach a final volume of 20 µL and the reaction mixture was incubated at for 1h at 37ºC. For chemical deglycosylation,analysis of secreted hIDUA, 1.5 mL media from four day old cultures was freeze dried after the overnight dialysis (Spectra/Por1 Dialysis membrane 12–14,000) in double-distilled water. Chemical deglycosylation was done by using the chemical deglycosylation kit GlycoProfile IV. The deglycosylated proteins were later dialyzed with double-distilled water and freeze-dried, followed by separation of protein via SDS-PAGE.
- Genomic DNA PCR
The genomic DNA of genetically modified BY-2 cell lines was extracted with the usage of CTAB buffer (2%, w/v) CTAB, 1.4 M NaCl, 20 mM Na2EDTA, 100 mM Tris–HCl, 0.2% (v/v) 2-mercaptoethanol, pH 8.0). The analogous hIDUA fragment for the verification of PCT was spliced using hIDUA-specific primers 5’-GGGGGATCCGAGGCCCCGCACCTGGTGCAG-3’ and 5’ GGGCTCGAGTCACAGTAGCAGGTTCTGATGCTGCGC- 3’.
- Subcellular localization
Fixation, preparation and immunolabeling of WT and transgenic tobacco BY-2 cells expressing SPp-IDUA for confocal immunofluorescence microscopy was done. For high-pressure freezing, BY-2 cells were accumulated by filtering and then immediately frozen in a high-pressure freezing apparatus. For subsequent freeze substitution, the frozen samples were initially kept at -85ºC for 60h, then eventually warmed over 18h at 0ºC. Substitution was carried out in an AFS freeze-substitution unit (Leica). The substitution medium (dry acetone) was supplemented with 0.1% (w/v) uranyl acetate. The medium was replaced with 100% ethanol when the samples reached 0ºC, which was further changed to freshly made 100% ethanol 10 min later. The cells were then infiltrated accordingly at -20ºC with HM20, embedded, and polymerized under UV light. Immunolabeling of HM20 sections was done using normal procedures; IDUA antibodies were used at a final concentration of 40 µg/mL and gold-coupled secondary antibodies were used at a 1:50 dilution. Lead citrate/aqueous uranyl acetate post-stained sections were analysed with the help of a Hitachi H-7650 transmission electron microscope which was equipped with a CCD camera operating at 80 kV.
- Determination of hIDUA activities
The culture media of genetically modified tobacco BY-2 cell lines were used directly in assays to determine IDUA working using the fluorogenic substrate sodium 4-methylumbelliferyl-a-L-iduronide (4MUI). 10 µL of substrate solution which consisted 0.1 M sodium dimethylglutarate buffer pH 4.5, 1 mM sodium metabisulphite, 3.5 mg/mL BSA and 0.75 mM 4MUI were added to 5 µL extract of protein and at 37ºC was incubated.Later the mixtures were incubated for a time period of 0, 30 and 60 min to have a confirmed linear rate of 4MUI production over the period of period. Samples were wrapped in foil to decrease the destruction of the light-sensitive substrate. Parallel substrate and extraction buffer blank samples were used as controls. The reaction was ended by the addition of 1.4 mL stop buffer which consisted of 0.2 M glycine, 0.125 M sodium carbonate, pH 10.7. Fluorescence of the reaction product, 4MU, was confirmed by the help of a fluorometer .
We have determined that a plant signal peptide sequence was more efficient for proper expression and discharge of recombinant human IDUA in genetically modified tobacco BY-2 cells. The production and working of hIDUA were evaluated to be around 53,000 pmol/min/mg protein and 10 mg hIDUA per mL of cultured media from long-term cultures of genetically modified tobacco BY-2 cells, which is equivalent to, or higher than those in genetically modified Arabidopsis cgl seeds or genetically modified CHO cells . The elevated activity of BY-2 cell-derived recombinant hIDUA demonstrates that this human enzyme with a plant signal peptide is appropriately processed, folded and focused for secretion. The existence of secreted hIDUA in cultured media of genetically modified BY-2 cells will take into consideration more advantageous and monetary downstream refinement of this enzyme, and therefore can possibly produce more affordable recombinant hIDUA to be utilized as a part of enzyme replacement therapy. Also, the present expression and secretion system utilizing tobacco BY-2 cells constitutes a helpful stage for the creation and purging of many more lysosomal enzymes in humans for the replacement therapy of enzymes and also many more different pharmaceuticals. (1)
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α-mannosidase in transgenic tobacco plants(Example 2):
α -Mannosidosis is an uncommon lysosomal storage disease because of continuous build up of undegraded oligosaccharides inside lysosomes. The insufficiency of the α -mannosidase enzyme is the reason for the ailment. The enzyme is made as a single chain precursor and sorted to the lysosomes where it is prepared. (2)
Two unique constructs were created: in the first, the full-length cDNA coding arrangement of
α-mannosidase(LAMAN) was under the control of the rubisco small subunit promoter and nopaline synthase terminator. In the second one, pGreen-LAMAN, the α -mannosidase cDNA expression was controlled by the CaMV 35S promoter and terminator. Also, the first 49 N-terminal signal peptide was replaced by a particular plant signal peptide from PR1 protein and the FLAG epitope was included at the C-terminus of the protein. Just the pGreen-LAMAN tobacco changed plants expressed the α -mannosidase enzyme. The major signal revelaed in Western blot experiments utilizing the anti-FLAG antibody had an atomic mass of around 110 kDa comparing to the whole protein precursor, demonstrating that the protein was effectively integrated. Western blot experiments utilizing antibodies particular for the α -mannosidase enzyme uncovered a few signals corresponding to the single chain precursor and glycopeptides derived from precursor proteolysis. Reconstructed plants expressing the protein displayed an enzymatic movement particularly higher then the unchanged tobacco plants. The recombinant enzyme demonstrated biochemical components practically identical to those of the human enzyme. (2)
1. Production and characterization of soluble human lysosomal enzyme. Lai Hong Fu, Yansong Miao, Sze Wan Lo , Tai Chi Seto , Samuel S.M. Sun , Zeng-Fu Xu. 2009.
2. Enzyme replacement therapy: Production of human a-mannosidase in transgenic tobacco plants. F. De Marchis, C. Balducci, R. Pagiotti , S. Arcioni , T. Beccari. 2010.