Biochemical characterization of the recombinant human Nogo-A ectodomain.

Nogo-A is a physiologically relevant inhibitor of neuronal growth and regeneration in the myelin of the adult human central nervous system and has attracted considerable attention as a molecular target for the treatment of spinal cord injuries. To gain insight into the structural and functional properties of the large extramembrane region that is characteristic for the Nogo-A splice form of this member of the Reticulon family of membrane proteins, we cloned and expressed the region comprising residues 334-966 as a soluble homogeneous protein in the periplasm of Escherichia coli. SDS/PAGE, under nonreducing conditions, and a systematic substitution analysis of all six Cys residues of Nogo-A indicated that this domain forms two structural disulfide bonds among Cys residues 424, 464, 559 and 597, whereas the Cys residues at positions 699 and 912 seem to be dispensable for folding.

Genetic design of an optimized packaging cell line for gene vectors transducing human B cells.

Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human gamma-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells.

Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701.

The orf virus (OV) strain D1701 belongs to the genetically heterogenous parapoxvirus (PPV) genus of the family Poxviridae. The attenuated OV D1701 has been licensed as a live vaccine against contagious ecthyma in sheep. Detailed knowledge on the genetic structure and organization of this PPV vaccine strain is an important prerequisite to reveal possible genetic mechanisms of PPV attenuation.