Article

Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

Center for Microbial Biotechnology, Institute of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
PLoS ONE (Impact Factor: 3.73). 01/2011; 6(6):e21267. DOI: 10.1371/journal.pone.0021267
Source: PubMed

ABSTRACT Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.

0 Bookmarks
 · 
147 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase A(1) (PLA(1)) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of Protein Kinase C. In the present work we determined higher levels of PLA(1) expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA(1) activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA(1) antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA(1) in the early events of parasite-host cell interaction. We also demonstrated the presence of Lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA(1) action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA(1) with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA(1) selected gene was cloned and expressed as a recombinant protein that possessed PLA(1) activity. Collectively, the results presented here point out at T. cruzi PLA(1) as a novel virulence factor implicated in parasite invasion.
    Molecular and Biochemical Parasitology 12/2012; · 2.73 Impact Factor

Full-text (2 Sources)

View
33 Downloads
Available from
May 21, 2014