Publikationsserver der RWTH Aachen University
Taraxacum officinale as an expression system for recombinant proteins : molecular cloning and functional analysis of the genes encoding the major latex proteins
Abstract
dc:descriptionThe latex from Dandelion, Taraxacum officinale Web., contains mainly four abundant peptides with an apparent molecular mass of 66, 60, 37, 18 kDa. These four peptides were termed major latex protein (MLPs). The identification of the MLPs and cloning of their corresponding genes were undertaken with the objective to isolate useful promoters to drive high level transgene expression in genetically engineered T. officinale plants, which would allow a cost-effective purification of recombinant proteins from latex. After determining the partial amino-acid sequences of the MLP cyanobromide peptide fragments, we reported that three MLPs (MLP66, MLP37 and MLP18) show high sequence homology to plant polyphenol oxidases (PPO). Based on these peptide fragments, an inverse PCR strategy was developed to amplify a unique sequence, encoding the latex PPO. RT-PCR was used to confirm the expression of the latex ppo gene and revealed that two closely related genes encoding a PPO are expressed in the latex. Furthermore, several microscopy techniques were developed to identify and analyse the laticifer organisation in T. officinale. The in vitro data suggested that PPO activity is exclusively responsible for the L-DOPA-oxidation observed in the laticifers and in the latex fractions. In addition, we produced and used polyclonal antibodies against the mature PPO form and against the PPO carboxyl domain to probe protein gel blots of latex and plant tissues from T. officinale. Immuno-blot experiments confirmed that MLP66, MLP37 and MLP18 belong to the same gene products and were generated after catalytic cleavage of a pre-mature PPO precursor (MLP66). The presence of three PPO forms in plant sample and the demonstration of catalytic cleavage of the PPO were shown for the first time in this thesis. The promoter region of the latex ppo gene was isolated by adaptor-anchored PCR and evaluated for high level expression of foreign protein in the latex. In this study, I present the results conducted in transgenic T. officinale and demonstrate that the ppo promoter was functional in the host plant. In transgenic T. officinale plants containing the ppo promoter region fused to the beta-glucuronidase (GUS) gene. GUS activity was restricted to the laticifers. Expression level was 50–fold higher in the laticifers than the expression level conferred by the CaMV 35S promoter in the same tissue. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the GUS expression profiles observed in T. officinale. Finally, the ability of T. officinale to express complex recombinant proteins in the latex was tested by expressing the HIV-specific full length 2G12 antibody. In the present study, we demonstrated the expression and assembly of full-length heavy and light chains to form functional 2G12 antibodies in T. officinale leaves and latex fraction.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2007
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Foucu, Florence
- Contributors dc:contributor
-
- Fischer, Rainer
Subjects
dc:subject × 9Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:62278