Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity.
Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star (DE3) cells was induced by the addition of 0.5mM IPTG. Cultures were grown at 24 and 37 degrees C, and VPr1 more readily partitioned into the soluble fraction at 24 degrees C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.
Dani
M Paulina
MP
Food and Environment Research Agency, Sand Hutton, York Y041 1LZ, UK. paulina.dani@fera.gsi.gov.uk
Richards
Elaine H
EH
D000818:Animals; D003001:Cloning, Molecular; D006434:Hemocytes / Q000187:drug effects / Q000276:immunology; D019476:Insect Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D009036:Moths / Q000276:immunology* / Q000469:parasitology; D014862:Wasp Venoms / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D014863:Wasps / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D019476:Insect Proteins; D014862:Wasp Venoms