High Affinity Human Antibody Fragments to Dengue Virus Non-Structural Protein 3

Dengue virus is the most prevalent mosquito transmitted infectious disease in humans and is responsible for febrile disease such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue non-structural protein 3 (NS3) is an essential, multifunctional, viral enzyme with two distinct domains; a protease domain required for processing of the viral polyprotein, and a helicase domain required for replication of the viral genome. In this study ten unique human antibody fragments (Fab) that specifically bind dengue NS3 were isolated from a diverse library of Fab clones using phage display technology. The binding site of one of these antibodies, Fab 3F8, has been precisely mapped to the third α-helix within subdomain III of the helicase domain (amino acids 526–531). The antibody inhibits the helicase activity of NS3 in biochemical assays and reduces DENV replication in human embryonic kidney cells. The antibody is a valuable tool for studying dengue replication mechanisms

Posttranslational processing of barley beta-glucan endohydrolases in the baculovirus insect cell expression system

Two cDNAs encoding barley (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes EI and EII have been expressed in Spodoptera frugiperda (Sf9) cell cultures using the baculovirus AcNPV vector. Modifications to both the 5′ and 3′ ends of the cDNAs were required before satisfactory levels of expression were obtained. The modified cDNAs directed high levels of (1→3,1→4)-β-glucanase expression in the Sf9 insect cell cultures, with yields of 10 mg/liter of isoenzyme EI (expEI) and 15 mg/liter of isoenzyme EII (expEII). Amino acid sequence analyses showed that the expressed enzymes were processed correctly at their amino termini. However, affinity chromatography of the isoenzyme expEII on concanavalin-A (conA)-Sepharose indicated that, although the enzyme is glycosylated, the structures of the carbohydrate chains differ from those of the native enzyme. When a cDNA encoding the homologous barley (1→3)-β-glucanase (EC 3.2.1.39) isoenzyme GII was expressed in insect cells, aberrant amino-terminal processing of the nascent polypeptide was sometimes observed. The forms with incompletely removed signal peptides retained their substrate specificity, but exhibited slightly reduced catalytic efficiency, altered Chromatographic behavior, and reduced stability at elevated temperatures. The results show that high levels of expression of recombinant plant proteins can be obtained in insect cells, but they emphasize the need to characterize thoroughly the products that are expressed in the heterologous insect cell system before comparisons are made with the native enzyme or with engineered enzyme mutants

Structure-guided fragment-based in silico drug design of dengue protease inhibitors

10.1007/s10822-011-9418-0Journal of Computer-Aided Molecular Design253263-274JCAD