Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom

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• 作者：Adriano M. Morgon^a ; Matheus R. Belisario-Ferrari^a ; Dilza Trevisan-Silva^a ; Gabriel O. Meissner^a ; Larissa Vuitika^a ; Brenda Marin^a ; Alexandre K. Tashima^d ; Luiza H. Gremski^a ; ^b ; Waldemiro Gremski^a ; ^c ; Andrea Senff-Ribeiro^a ; Silvio S. Veiga^a ; Olga M. Chaim^a ; ^{olgachaim@ufpr.br" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Loxosceles intermedia ; Venom ; Astacin ; Metalloprotease ; Toxin ; Recombinant protein
• 刊名：Biochimie
• 出版年：2016
• 出版时间：September-October 2016
• 年：2016
• 卷：128-129
• 期：Complete
• 页码：8-19
• 全文大小：2480 K

文摘

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2–40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.