کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6490108 | 1416960 | 2018 | 56 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis
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کلمات کلیدی
RAMOSOTRUstilago maydisGlcHRPIMACMCsGUSCBBCarboxypeptidaseβ-glucuronidase - β-گلوکورونیدازProtein expression - بیان پروتئینUnconventional secretion - ترشح غیر متعارفShh - خیرmultiple cloning site - سایت کلونینگ چندگانهoxygen transfer rate - سرعت انتقال اکسیژنhorse radish peroxidase - پراکسیداز تربچه اسبcomplete medium - کامل رسانهimmobilized metal ion affinity chromatography - کروماتوگرافی جذب یون فلز بی حرکتیGlucose - گلوکز
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The eukaryotic microorganism Ustilago maydis is currently being developed as an alternative protein expression platform. Protein fusion with an unconventionally secreted chitinase mediates export of heterologous proteins. The unique feature of this pathway is the circumvention of N-glycosylation. Different heterologous proteins could already be secreted via this novel mechanism in their active state. However, the system still suffers from low yields mainly attributed to the degradation of exported recombinant proteins by proteases. Here, we combined optimization steps on the level of cultivation conditions and strain engineering to further improve the system. Using the Respiration Activity Monitoring System we discovered that a pH drop during prolonged incubation results in loss of activity and degradation of the target protein. This problem can be reduced by buffering the cultivation medium. However, we still observed significant proteolysis even in buffered cultures. Hence, we revisited strain engineering to reduce the proteolytic activity. Secreted proteases were discovered using mass spectrometry. Then, genes for three identified proteases of a serine-carboxypeptidase family were deleted in an existing quintuple protease deletion mutant. This further diminished proteolytic activity and target protein degradation. The two approaches overall strongly improved the stability of heterologous proteins in this fungal system.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 284, 20 October 2018, Pages 37-51
Journal: Journal of Biotechnology - Volume 284, 20 October 2018, Pages 37-51
نویسندگان
Marius Terfrüchte, Sandra Wewetzer, Parveen Sarkari, Daniel Stollewerk, Mirita Franz-Wachtel, Boris Macek, Tino Schlepütz, Michael Feldbrügge, Jochen Büchs, Kerstin Schipper,