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Mayer, Florian (2012): The expression of CPP fusion proteins in plastids. Dissertation, LMU München: Faculty of Biology
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Abstract

Plastid transformation is a valuable technique for both basic and applied science. In basic science the technique is used to study chloroplast function. Applied approaches deal with the potential of the plastid for the production of medicinal therapeutics, in most cases vaccine antigens coupled to adjuvants. Adjuvants are used for the trans-mucosal delivery of attached cargoes. Cell penetrating peptides (CPPs) emerged as valuable tools for the delivery of cell-impermeable cargoes across cell barriers more than twenty years ago. Although the exact mechanism of CPP penetration of cells is still discussed, the applied value of CPPs is documented in a number of clinical studies. Recently, scientists working in the CPP field launched a call for an alternative expression platform for CPP fusion peptides / proteins. Only a short time before, CPPs were introduced into plant science and some impressive first results, manipulating plant cells from the “outside”, were achieved. The present study aimed at combining the fields of plastid transformation and CPPs from the “inside”. We report the first expression of CPP fusion proteins in a plant, more precisely in the plastid. The approach focused on three aspects of CPP fusion protein expression in the organelle: (A) the principal feasibility of CPP-fusion protein expression in the plastid, (B) the location of CPP fusion proteins in the plant cell upon plastid-based expression and (C) the use of plastids for the manufacture of CPP fusions to provide an alternative to the bacterial expression system. Nine prominent CPPs were employed in three vector series to investigate these aspects. In vector series I the selected CPPs were fused to the fluorescent protein eGFP to provide an optical read-out; in vector series II, the CPPs were fused to Arabidopsis MYB transcription factor PAP1 to provide a biological read-out and in vector series III, two CPPs were fused to the human enzyme PAH to introduce plant-based CPP fusion protein expression. Taken together, the expression of CPP fusion in the plastid turned out to be feasible. Transplastomic plants reached homoplasmy, produced viable seeds and stably inherited the desired trait to their progenies in a maternal fashion. Only low protein accumulation levels were detected. Pleiotropic effects occured at the low protein accumulation levels observed. Localisation of CPP fusion proteins was shown to be restricted to the plastid. An inability of CPP fusion proteins isolated from vector series I to penetrate protoplasts, young plant tissue and human cell lines was revealed. The value of a plastid-based manufacture of CPP fusion proteins for clinical approaches failed to be demonstrated due to low fusion protein accumulation levels. Bottlenecks of the current study are discussed and suggestions are made to provide a framework for future efforts.