Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

Immunolocalization of the saposin-like insert of plant aspartic proteinases exhibiting saposin C activity. Expression in young flower tissues and in barley seeds

The plant- specific insert ( PSI) of cypro11 gene- encoding cyprosin, an aspartic proteinase from Cynara cardunculus, has been cloned by polymerase chain reaction ( PCR) into a bacterial expression vector. A rearranged form of this PSI in which the N- and C- terminal sequences were permutated to make it more similar to the structural arrangement observed in saposins was also cloned and expressed in the same system. The biological activities of the two purified recombinant proteins were compared to those of human saposins B and C. The proteins showed similar activity to saposin C, i. e. capacity to activate human glucosylceramidase. At a concentration of 5 mu M, wild- type PSI, saposin C, and rearranged PSI activated human glucosylceramidase two-, three-, and five- fold, respectively. The K-m for 4- methylumbelliferyl beta-glucopyranoside was around 7 mM in the presence of any of the three activators ( 5 mM). The neurotropic activity using NS20Y cells and lipid- binding properties of the plant recombinant proteins were tested. The two plant proteins showed lipid- binding properties similar to those of saposins but did not have any effect on neurite outgrowth. Immunolocalization of PSI showed its expression in protective tissues in flower meristem - protodermis, in C. cardunculus and embryonic root cap and coleorhiza in mature barley grains - as well as husk, pericarp, and the aleurone layer. Possible biological functions suggested for the plant homologue to saposins besides the general activation of enzymes involved in lipid metabolism would be involvement in plant defence

Amorpha-4,11-diene synthase: Mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate

Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-H-2]FPP, (1S)-[1-H-2]FPP, and [1,1-H-2(2)]FPP. GC-MS analysis of amorpha-4,11-diene formed from the deuterated FPPs shows that the deuterium atoms are retained in the product. Furthermore, analysis of the MS-spectra obtained with the differently labeled substrate indicates that the H-1si-proton of FPP is transferred during the cyclization reaction to carbon 10 of amorphadiene while the H-1re-proton of FPP is retained on C-6 of the product. Proton NMR and COSY experiments proved that the original H-1si-proton of FPP is located at C-10 of amorpha-4,11-diene as a result of a 1,3-hydride shift following initial 1,6-ring closure. The results obtained support the previously suggested mechanism for the cyclization of farnesyl diphosphate by amorph-4,11-diene synthase involving isomerization of FPP to (R)-nerolidyl diphosphate (NPP), ionization of NPP, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure to generate the amorphane skeleton, and deprotonation at either C-12 or C-13 to afford the final product (1S,6R,7R,10R)-amorpha-4,11-diene. (c) 2005 Elsevier Inc. All rights reserved

Molecular Cloning, Expression, and Characterization of Amorpha-4,11-diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L

In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5′-untranslated end, and a 427-bp 3′-untranslated sequence. The deduced amino acid sequence is 32 to 51␒dentical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5€and oxygenated (2.5€sesquiterpenes from farnesyl diphosphate. GC–MS analysis identified the olefins as (E)--farnesene (0.8Œ amorpha-4,11-diene (91.2Œ amorpha-4,7(11)-diene (3.7Œ -humulene (1.0Œ -sesquiphellandrene (0.5Œ and an unknown olefin (0.2€and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2€(tentatively), amorpha-4-en-7-ol (2.1Œ and -bisabolol (0.3€(tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2 , and Mn2 are 0.9, 70, and 13 M, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested