کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5509166 | 1538403 | 2016 | 41 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom](/preview/png/5509166.png)
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimie - Volumes 128â129, SeptemberâOctober 2016, Pages 8-19
Journal: Biochimie - Volumes 128â129, SeptemberâOctober 2016, Pages 8-19
نویسندگان
Adriano M. Morgon, Matheus R. Belisario-Ferrari, Dilza Trevisan-Silva, Gabriel O. Meissner, Larissa Vuitika, Brenda Marin, Alexandre K. Tashima, Luiza H. Gremski, Waldemiro Gremski, Andrea Senff-Ribeiro, Silvio S. Veiga, Olga M. Chaim,