Purification of Leptinotarsa decemlineata (Say) Gut Specific Cysteine Protease Inhibitor(s) From Rapeseed
Abstract:
The aim of the present work was to purify cysteine protease inhibitors from rapeseed (Brassica napus L.), with potential activity on digestive protease of Colorado Potato Beetle (CPB), Leptinotarsa decemlineata (Say). Ammonium sulfate precipitated proteinaceous fractions; 30, 50, 70, and 100% showed 39.07, 57.03, 51.47, and 22.44% inhibition on the fourth instar larval gut general protease activity, respectively. Fraction 50% showed the highest inhibitory effect on digestive general protease activity of all developmental stages. Gel assays approved the inhibition of the enzyme activity. Fraction 50% was purified by using various chromatography techniques; ion-exchange using DEAE, gel filtration and affinity using SiO2-CPB larval gut homogenate. Three peaks of protein were eluted from ion exchange chromatography using NaCl step gradient, also from gel filtration chromatography. When Z-Ala-Arg-Arg-4mßNA was used as cysteine protease substrate, the purification fold of second fraction of ion exchange chromatography was obtained 24.80, also the yield was 59.09%, the third fraction of gel permeation resulted in a 25.60 fold purification with 28.53% of recovery, and the fraction of affinity chromatography obtained a 22.72 fold purification and yielded 36.35%. In the SDS-PAGE, apparent molecular mass of purified proteins were 34 and 32 kDa by ion-exchange and 24 and 22 kDa by affinity. However, gel filtration was not an appropriate method in this study, because the purified protein band(s) were not observed on the gel. Consequently, these chromatography methods were appropriate methods to purification of inhibitor cystatins, specially affinity which was prepared by using CPB gut enzyme as ligand and obtained specific inhibitor proteins of CPB gut protease activity.
Language:
English
Published:
Journal of Agricultural Science and Technology, Volume:19 Issue: 3, May 2017
Pages:
653 to 667
magiran.com/p1687485  
دانلود و مطالعه متن این مقاله با یکی از روشهای زیر امکان پذیر است:
اشتراک شخصی
با عضویت و پرداخت آنلاین حق اشتراک یک‌ساله به مبلغ 990,000ريال می‌توانید 70 عنوان مطلب دانلود کنید!
اشتراک سازمانی
به کتابخانه دانشگاه یا محل کار خود پیشنهاد کنید تا اشتراک سازمانی این پایگاه را برای دسترسی نامحدود همه کاربران به متن مطالب تهیه نمایند!
توجه!
  • حق عضویت دریافتی صرف حمایت از نشریات عضو و نگهداری، تکمیل و توسعه مگیران می‌شود.
  • پرداخت حق اشتراک و دانلود مقالات اجازه بازنشر آن در سایر رسانه‌های چاپی و دیجیتال را به کاربر نمی‌دهد.
دسترسی سراسری کاربران دانشگاه پیام نور!
اعضای هیئت علمی و دانشجویان دانشگاه پیام نور در سراسر کشور، در صورت ثبت نام با ایمیل دانشگاهی، تا پایان فروردین ماه 1403 به مقالات سایت دسترسی خواهند داشت!
In order to view content subscription is required

Personal subscription
Subscribe magiran.com for 50 € euros via PayPal and download 70 articles during a year.
Organization subscription
Please contact us to subscribe your university or library for unlimited access!