Plantibodies : requirements for expression and subcellular targeting

Research output: Thesisinternal PhD, WU

Abstract

<p>Engineering resistance against pathogens is often frustrated by the lack of suitable genes. This problem can be overcome by <em>in planta</em> expression of antibodies (plantibodies) able to inactivate pathogen specific proteins. Since antibodies can be generated against almost any molecular structure, this strategy should be feasible for various diseases and pests.</p><p>Antibodies have several desirable features with regard to protein engineering. Using molecular techniques the relatively large antibody molecule (150 kDa), consisting of two heavy and two light chain molecules, can be trimmed to a single-chain antibody fragment (scFv) of 30 kDa, which consists of a variable heavy and light domain connected by a linker peptide.</p><p>This thesis describes the expression and subcellular targeting of functional full size antibodies and various types of scFv fragments in tobacco plants. The light and heavy chain coding genes of a full size model antibody were efficiently introduced in tobacco plants. A large number of the transformants expressed and secreted relatively high levels of the functional antibody.</p><p>From the model antibody an scFv fragment was derived and expressed in tobacco. The scFv was poorly secreted. However, the scFv fragments could be expressed intracellularly to high protein levels in the endoplasmic reticulum (ER) by adding the KDEL retention signal, and in the ectopic environment of the cytosol by omitting the ER translocation signal. Remarkably, the cytosolic scFv could only be expressed at detectable levels when the KDEL sequence was present. Immunoelectron microscopy and biochemical markers confirmed the proper subcellular locations of these intracellularly expressed scFv fragments. The KDEL extension also improved cytosolic expression levels of several other scFv fragments.</p><p>The <em>in vivo</em> redox state of these intracellularly expressed scFvs was determined. Both the variable heavy and light domains contain a disulfide bridge, which is considered crucial for stability and functionality of the scFv. As expected, these disulfide bridges were present in the oxidizing environment of the ER. Remarkably, in stable transformants the intramolecular disulfide bridges were also found in the scFvs present in the reducing environment of the plant cytosol, resulting in a functional antibody fragment. Like in the ER, the formation of intermolecular disulfide bridges was also observed, which resulted in functional dimers. When transiently expressed in the cytosol, the scFv was present in a reduced and non-functional form, confirming that the intramolecular disulfide bridges are necessary for the functionality.</p><p>In an attempt to improve scFv secretion levels, the model scFv was modified without changing the variable domains. The linker peptide was changed and modified, and expression levels were compared in transient expression assays. This only influenced the intracellular accumulation indicating that the secretory scFv was probably degraded somewhere in the secretory pathway. Various novel types of dimerized scFvs, by adding an extra domain derived from camel or IgM antibodies, were expressed transiently and in stable transformants. The addition of the camel IgG <sub>2</sub> 'long hinge' in combination with the IgM C <sub>H</sub> 4 domain did significantly improve secretion.</p><p>It can be concluded that scFvs, unlike full size antibodies, can be targeted to various subcellular compartments. This allows direction of scFv fragments to locations where the pathogen specific protein is present. This thesis demonstrates that antibodies are versatile molecules and that engineering resistance through plantibody technology against nematodes, fungi and insects seems within reach.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Bakker, Jaap, Promotor
  • Schots, Arjen, Promotor
  • Gommers, F.J., Promotor, External person
Award date30 Oct 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054859161
Publication statusPublished - 1998

Keywords

  • disease resistance
  • antibodies
  • plants
  • genetic engineering
  • plantibodies

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