Cloning and expression of the gene for an insect haemocyte anti-aggregation protein (VPr3), from the venom of the endoparasitic wasp, Pimpla hypochondriaca.
A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti-aggregation activity in vitro and shares the same N-terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full-length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N-terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1 mM) and growth of the bacteria at 37 degrees C for 5 h, or at 24 degrees C for 20 h. Following lysis of bacteria grown at 37 degrees C, the target protein partitioned into the insoluble fraction. However, at 24 degrees C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24 degrees C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro.
Dani
M Paulina
MP
The Food and Environment Research Agency, Sand Hutton, York, United Kingdom. paulina.dani@fera.gsi.gov.uk
Richards
Elaine H
EH
D000818:Animals; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000187:drug effects; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006434:Hemocytes / Q000187:drug effects; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D009036:Moths / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D012995:Solubility; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism*; D014863:Wasps / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D019476:Insect Proteins; D014862:Wasp Venoms