oai:publications.rwth-aachen.de:62243
type: CreativeWork
name: 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
description: 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 fra…
abstract: 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 fra…
author: Nölke, Greta
language: eng
keywords: info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, polyamine manipulation, scFvs, transgenic plants
date published: 2002
date modified: 2026-07-30T19:43:19Z
provider: RWTH Aachen University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Publikationsserver der RWTH Aachen University