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
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis
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
نویسندگان
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