Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

Abstract

Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

Short Communication Characterization of chloroplast region rrn16-rrn23S from the tropical timber tree Cedrela odorata L. and de novo construction of a transplastomic expression vector suitable for Meliaceae trees and other economically important crops

“…The rrn16-rrn23 sequence showed considerable differences from the N. tabacum plastome. Previous reports (Gao et al, 2012;Rigano et al, 2012) suggest that a lack of homology in flanking regions may severely affect the recombination rate if the chloroplast transformations of C. odorata and other Meliaceae trees used previously reported vectors based on the N. tabacum plastome sequence. Recombination efficiency is the first of 2 widely recognized bottlenecks for a successful transplastomic plant regeneration protocol; the regeneration protocol is the second relevant limitation.…”

7. Themenbereich grüne Gentechnologie: Pflanzenzüchtung und Agrarwirtschaft

“…5. Ein weiterer Vorteil liegt darin, dass die für die Transformation eingesetzte DNA nicht zufällig in das Plastidengenom (Plastom) integriert wird, sondern mittels homologer Rekombination (Rekombination an DNA-Stellen mit ähnlicher DNASequenz), sodass der Integrationsort bekannt ist und unbeabsichtigte Positionseffekte reduziert werden (Day/Goldschmidt-Clermont, 2011;Gao et al, 2012).…”

“…Inzwischen ist sie zu einer Technologie mit unterschiedlichen Anwendungen gereift. 20 Bis heute sind bereits mehr als 50 verschiedene Transgene stabil in Plastidengenome integriert worden, weiterhin jedoch ist die gentechnische Modifikation der Plastidengenome von monokotylen Pflanzen, zu denen die wichtigen Getreidepflanzen wie beispielsweise Mais, Reis oder Weizen gehören, nicht möglich (Gao et al, 2012;Bock, 2014). Die Transplastomtechnologie wird unter anderem für die Produktion von pharmakologisch relevanten Proteinen PMPs,vgl.…”

“…Aufgrund von toxischen Alkaloiden im Tabak eignet er sich jedoch nicht für die orale Impfung. Kommerzielle Anwendungen der Plastidentransformation sind bislang noch in der Entwicklung, ein kommerzieller Anbau solcher Pflanzen erfolgt derzeit nicht (Maliga/Bock, 2011;Gao et al, 2012;Bock, 2014).…”

Rapid production of therapeutic proteins using plant system

“…Since plants have long been used for the production of many protein therapeutics, there are many shortcomings affect the plant produced protein therapeutics to meet commercialisation standards. Recent developments in the plant molecular farming especially the development of simplest protein purification strategies, deconstructed viral vectors, glyco engineering and magnifection technology 12,[14][15][16][17][18][19][20][21][22][23] were the most astonishing developments added more value to the plant-based therapeutics that are already commercialised and in various phases of clinical trials.…”