Direct fluorometric measurement of hepatitis C virus helicase activity.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established.

Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm.

A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag.

Expression and one-step purification of intracellular human prolactin in insect cells.

Human prolactin was expressed in insect culture cells by recombinant baculoviruses carrying prolactin gene cDNA placed under the transcriptional control of polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus. Preliminary results of recombinant human prolactin expression as extracellular as well as intracellular product of baculovirus expression system were presented at the FEBS Meeting in Nice, France, in 1999 (Abstracts, p. 288).

Genetic variability of four natural isolates of the Stilpnotia salicis multiple-enveloped nuclear polyhedrosis virus.

Viral genome DNA from four different multiple-enveloped nuclear polyhedrosis virus isolates, obtained from naturally infected larvae of satin moth (Stilpnotia salicis), a pest of poplar tree (Populus) was analysed. Larvae were collected over a period of 11 years, from 1978 to 1989. The genomic DNA restriction patterns pointed to heterogeneity of these wild-type viruses.