ABSTRACT
An effective and affordable treatment against malaria is still a challenge for medicine. Most contemporary drugs either are too expensive to produce or are not effective against resistant strains of the malaria parasite Plasmodium falciparum. The plant Artemisia annua L. is the source of artemisinin, an effective drug against malaria for which no resistant strains of the bacterium have been reported. However, the artemisinin content of A. annua is very low, which makes its production expensive. Here we report the use of transgenic technology to increase the artemisinin content of A. annua. We report the production of transgenic plants of A. annua into which we transferred 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) gene from Catharanthus roseus (L.) G. Don using Agrobacterium-mediated gene transfer technology. Transgene integration and copy number were assessed by PCR and Southern hybridization, which confirmed the stable integration of multiple copies of the transgene in 7 different transgenic lines of A. annua. The leaf tissue of three of the A. annua transgenic lines possessed significantly higher HMGR activity compared with wild-type controls, and this activity was associated exclusively with microsomal membranes. The artemisinin content of the shoots of one of the transgenic lines depicted an increase of 22.5 % artemisinin content compared with wild-type control A. annua plants.
Subject(s)
Artemisia annua/genetics , Artemisinins/metabolism , Gene Expression , Genes, Plant/physiology , Hydroxymethylglutaryl CoA Reductases/genetics , Plant Extracts/metabolism , Antimalarials/metabolism , Artemisia annua/metabolism , Blotting, Southern , Catharanthus/genetics , Hydroxymethylglutaryl CoA Reductases/metabolism , Intracellular Membranes , Microsomes , Plant Extracts/genetics , Plant Leaves , Plants, Genetically Modified , Polymerase Chain Reaction , RhizobiumABSTRACT
The maltose binding protein (MBP) affinity tag has been extensively used for protein purification. A commercial grade cationic starch could precipitate MBP or an MBP-tagged protein quantitatively by simultaneous addition of 10% (w/v) polyethylene glycol (PEG) and 50 mM calcium chloride. The precipitated MBP or MBP-tagged protein could be selectively dissociated by suspending the precipitate in 1 M NaCl. In the case of a soluble MBP fusion with a fragment of human immunodeficiency virus protein gp120, 38% of the contaminating proteins could be removed by precipitation with PEG/CaCl(2) and 100% of the fusion protein was recovered. In all cases, the purified proteins showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expected changes in fluorescence emission spectra upon binding to maltose.
