Expression and processing of cecropin and attacin using a baculovirus vector

Sammanfattning: The cecropins and attacins are two antibacterial peptides induced uponimmunization in the moth Hyalophora cecropia. They are both synthesized aspreproproteins that are processed and post-translationally modified along the exportpathway.To study these processing and modification events, the baculovirus expressionvector system was employed as a model system. The strong polyhedrin promoter ofAutographa californica nuclear polyhedrosis virus (AcNPV) was used to driveexpression of different forms of cecropin and attacin. To successfully express thesesmall proteins that are sensitive to proteolysis, Trichoplusia ni larvae and H.cecropia pupae were used instead of Spodoptera frugiperda cell culture. Therecombinant proteins produced by living animals were biologically active, accuratelyprocessed and the cecropins were C-terminally amidated.The amidation machinery of H. cecropia was further investigated. It was foundthat enzymes needed for amidation are present in small amounts in both fat body andhemolymph of H. cecropia pupae, and that a bacterial infection causes an increase ofthe amidation activity, indicating a coregulation of the immune proteins and theenzymes needed for their processing.In order to establish that T. ni was a suitable host for heterologous expression of theantibacterial peptides, the endogeneous immune response of the larvae wasinvestigated and it was ascertained that the immune proteins do not have a deleteriouseffect on AcNPV.The potential of diapausing H. cecropia pupae as expression host was explored byexpressing four different recombinant proteins. It was shown that H. cecropia pupaecompare favourably as expression host to T. ni larvae.