کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8359275 | 1542289 | 2018 | 30 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Downstream processing of a plant-derived malaria transmission-blocking vaccine candidate
ترجمه فارسی عنوان
فرایند فرآوری پایین از یک واکسن مسدود کننده مالاریا حاصل می شود
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کلمات کلیدی
mAbDOEAExDSPHCPNTUTSPMonoclonal antibody - آنتی بادی مونوکلونالstandard deviation - انحراف معیارUltrafiltration - اولترافیلتراسیونHIC - اینBlanching - بلانچینگFlow through - جریان از طریقDiafiltration - دیافیلتراسیونcolumn volume - ستون حجمDesign of experiments - طراحی آزمایشاتDownstream processing - فرایند فرآیند پایینAnion exchange - مبادله آنیونnephelometric turbidity unit - واحد کدورت غیرفلومتریکhost cell protein - پروتئین سلولی میزبانhydrophobic interaction chromatography - کروماتوگرافی تعامل هیدروفوبیtotal soluble protein - کل پروتئین محلول
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
چکیده انگلیسی
Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove â¼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of â¼75â¯Â°C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80â¯Â°C to 70â¯Â°C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of â¼72% with 60% recovery via step elution. We found that 30-75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9â¯mgâ¯kgâ1 plant material after purification based on an initial yield of â¼49â¯mgâ¯kgâ1 biomass after blanching.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 152, December 2018, Pages 122-130
Journal: Protein Expression and Purification - Volume 152, December 2018, Pages 122-130
نویسندگان
Stephan Menzel, Tanja Holland, Alexander Boes, Holger Spiegel, Rainer Fischer, Johannes Felix Buyel,