ABSTRACT
Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8 units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).
ABSTRACT
KEY MESSAGE: This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.
Subject(s)
Genetic Vectors , Nicotiana/genetics , Recombinant Proteins/metabolism , Agrobacterium/genetics , Cell Culture Techniques , Green Fluorescent Proteins , Luminescent Proteins/genetics , Plant Cells/metabolism , Plants, Genetically Modified/genetics , Protein Engineering/methods , Recombinant Proteins/genetics , Replicon , Nicotiana/cytology , Red Fluorescent ProteinABSTRACT
Porcine circovirus type 2 (PCV-2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus-associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV-2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV-2 capsid protein (CP) from plants is an essential first step towards the goal of a plant-produced PCV-2 vaccine candidate. In this study, the PCV-2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV-2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self-assembled into virus-like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant-produced PCV-2 VLPs elicited specific antibody responses to PCV-2 CP. This is the first report describing the expression of PCV-2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.
