Jim W Barnett

Human β nerve growth factor obtained from a baculovirus expression system has potent in vitro and in vivo neurotrophic activity

A baculovirus expression vector, which contains the coding sequences for human prepro (beta) nerve growth factor under control of the viral polyhedrin promoter, was constructed. Upon infection of insect cells with the recombinant virus, mature human beta nerve growth factor (rhNGF) was released into the culture fluid. The mature rhNGF was biologically active since rat pheochromocytoma (PC12) and human neuroblastoma (SH-SY5Y) cells were induced to extend neurites upon treatment with this material. This activity was abolished by treating with antiserum prepared against mature mouse beta NGF (mNGF). When compared with mNGF, rhNGF more rapidly elicited the differentiation response in both PC12 and SH-SY5Y cells. In an in vivo assay of cholinergic cell survival, rhNGF was nearly as potent as mNGF in protecting cholinergic neurons from degeneration following a fimbria-fornix lesion. These results show that the baculovirus expression system provides quantities of biologically potent human beta NGF suitable for a comprehensive program of research to ascertain beta NGFs potential as a therapeutic agent for the treatment of Alzheimers disease.

Physicochemical characterization of recombinant human nerve growth factor produced in insect cells with a baculovirus vector.

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion‐exchange and reversed‐phase chromatography to near homogeneity. The Nterminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (<0.08 mol of N‐acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF frominsect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimers disease.

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.