Engineering and characterization of single chain antibody fragments (scFvs) specific to key enzymes in polyamine biosynthesis and manipulation of polyamine pathway by constitutive expression of recombinant ODC and SDE enzymes in transgenic tobacco
Polyamines are ubiquitous low-molecular weight polycationic compounds found in all living organisms and involved in crucial developmental, physiological and metabolic processes. The majority of information available about the polyamine pathway was obtained by using a wide range of metabolic inhibitors (Evans and Malmberg, 1989; Tiburcio et al., 1990) and modulation of specific enzymatic activities via heterologous gene expression in transgenic plants. Previous studies have indicated that the polyamine pathway is tightly controlled in the end product accumulation. The objective of this study was to achieve changes in the pathway by combining genetic engineering and antibody technology approaches. ODC a key enzyme in the polyamine biosynthesis, was amplified from a human prostate cDNA library and subcloned into plant expression vector for transient expression in the apoplast, cytosol and endoplasmatic reticulum. ODC was accumulated to higher levels in plant cytosol and apoplast, while ODC targeted to ER was not detected in immunoblots. Stable transformed tobacco plants overexpressing heterolougs ODC in cytosol and apoplast were generated. Lines with the highest accumulation levels were selected for establishment of homozygous lines. The T2 generation of transgenic plants overexpressing human ODC in apoplast, ‘‘non-native compartment’’ of ODC revealed up to 32-fold elevated levels of ODC activity and accumulated significantly higher levels of putrescine. Additionaly, stable transformed tobacco plants co-expressing human ODC and chimeric human mouse SDE in apoplast were generated. Immunoblot analyses revealed that more than 80% of the lines tested co-expressed both enzymes. The best expressers showed significantly increased levels of putrescine (up to 5-fold) and spermidine (up to 4.5-fold). No increased levels of spermine were observed. The ADC, ODC and PAO key enzymes in polyamine pathway were chosen as a target for generation of specific scFv recombinant antibody fragments by phage display technology. Mice were immunized with the bacterial produced and affinity purified ADC, ODC-GST fusion protein and PAO-GST inclusion bodies. High antibody titers showed successful immunization. Three phage display libraries were constructed using the immunized mice repertoire. Recombinant antibodies were isolated by solid phase panning using bacterial ADC- and ODC-GST fusion proteins. Their specific reactivity to ADC and ODC was analysed by direct and capture ELISA. Sequence analyses revealed selection of two and four scFvs specific to ADC and ODC, respectively. Electromobility shift assays confirmed the functionality of the affinity purified scFvODC1 and scFvODC3. The binding properties of scFvODCs on soluble antigen were further verified by solution competition as monitored by surface plasmon resonance. The epitope specificity of selected scFvs was determined in ELISA and BIAcore assays. The pairwise epitope mapping indicated that scFvADC3 and scFvADC7 recognize independent epitopes in ADC, while scFvODC1 recognize a distinct epitope to scFvODC3, 7 and 15. Gel filtration assays indicated the presence of monomeric and dimeric forms of scFvODC3 and scFvODC7. In vitro assays indicated increase of the human ODC activity after addition of 3.4 mM of scFvODC1 and scFvODC3 of 75% and 92%, respectively. All selected scFvs were cloned into pTRA plant expression vector and analysed for their stability and accumulation levels in the plant cytosol, apoplast and ER. Immunoblot analyses of tobacco leaves demonstrated accumulation of scFvs in apoplast and ER with higher levels in ER. Moreover, scFvODC1 and scFvODC3 accumulated in high levels (up to 50-70 mg/g fresh weight) in the plant cytosol retaining the antigen binding specificity and activity in the reducing environment of the plant cytosol. Stable transformed plants expressing scFvODC1 and scFvODC3 specific to ODC in the apoplast and cytosol were generated. More than 67% of the regenerated plants expressed the apoplastic scFvODC1 and scFvODC3. The data presented in this study demonstrated changes in the polyamine pathway by constitutive expression of heterologous ODC and co-expression of ODC and SDE enzymes indicating that the polyamine pathway exhibit plasticity. Up to date this is the first study attempting to modulate the polyamine pathway by simultaneous expression of two enzymes involved in the polyamine synthesis. Moreover, generation of the scFvs specific to ADC and ODC key enzymes in the polyamine biosynthesis could be a valuable molecular tool for altering the pathway by using immunomodulation approaches and crossing experiments with the homozygous transgenic lines overexpressing ADC and/or ODC.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, polyamine manipulation, scFvs, transgenic plants
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