کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8359965 | 1542326 | 2015 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A2 from Neurospora crassa
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
PLA2His6NDSBNeurospora crassaPoPCsPLA2lysophosphatidylethanolamineLPELPCORFESI-MSphospholipase A2 - آنزیم فسفولیپاز A2 Electrospray Ionization Mass Spectrometry - اسپکترومتر جرم یونیزاسیون ElectrospraySecretory phospholipase A2 - اسید فسفولیپاز A2Refolding - بازپرداختsecretory PLA2 - ترشح PLA2circular dichroism - رنگ تابی دورانیphosphatidylcholine - فسفاتیدیل کولینopen reading frame - قاب خواندن بازLysophosphatidylcholine - لیزوفسفاتیدیل کولینhexahistidine - هگزاشیستیدینPOPE - پاپ
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca2+-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 115, November 2015, Pages 69-75
Journal: Protein Expression and Purification - Volume 115, November 2015, Pages 69-75
نویسندگان
Ayumi Takayanagi, Takuya Miyakawa, Atsuko Asano, Jun Ohtsuka, Masaru Tanokura, Manabu Arioka,