ABSTRACT
Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.
Subject(s)
Laccase/genetics , Laccase/isolation & purification , Polyporales/enzymology , Seeds/enzymology , Zea mays/genetics , Apoenzymes/isolation & purification , Apoenzymes/metabolism , Chlorides/metabolism , Copper/metabolism , Fungal Proteins/genetics , Laccase/biosynthesis , Lignin/metabolism , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Polyporales/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Zea mays/enzymologyABSTRACT
Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.
Subject(s)
Escherichia coli Proteins , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Vaccines, Synthetic/genetics , Animals , Bacterial Toxins/genetics , Bacterial Toxins/isolation & purification , Biotechnology , Enterotoxins/genetics , Enterotoxins/isolation & purification , Mice , Mice, Inbred BALB C , Plasmids/genetics , Rhizobium/genetics , Transformation, Genetic , Transmissible gastroenteritis virus/genetics , Transmissible gastroenteritis virus/immunology , Vaccines, Synthetic/isolation & purification , Viral Proteins/genetics , Viral Proteins/isolation & purification , Zea mays/geneticsABSTRACT
A sequence element within pBR322 DNA mediates a cis-acting negative effect on expression from eucaryotic genes in transient expression assays. The negative element overlaps with sequences that inhibit DNA replication, but its effect is observed in the absence of detectable replication of transfected DNA.
