کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1175704 961814 2006 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
In vivo optimizing of intracellular production of heterologous protein in Pichia pastoris by fluorescent scanning
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
In vivo optimizing of intracellular production of heterologous protein in Pichia pastoris by fluorescent scanning
چکیده انگلیسی

Specific monitoring of recombinant protein titer in DNA recombinant biotechnology traditionally has relied on SDS–PAGE, Western blotting, or bioactivity-based assays, but these are labor-intensive, time-consuming, and destructive and are not a good choice for the optimization of recombinant protein production. We describe a study in which enhanced green fluorescence protein (EGFP) was fused to the C terminus of a model protein glutathione S-transferase (GST) to optimize the chimeric protein production in Pichia pastoris by measurements of fluorescence of living cells in a 96-well microtiter plate using simple fluorescent scanning. Several common factors (e.g., time course of expression, effect of methanol concentration, frequency of methanol addition, medium pH) were tested using this strategy. Western blotting assay showed that the correct full-length GST–EGFP chimeric protein was expressed intracellularly in P. pastoris. The fluorescence intensity and GST bioactivity of cell extract yielded a direct correlation. The results show that the reported method provides an attractive platform for the optimization of recombinant protein production in vivo in real time as well as handling at least 96 samples in parallel.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 357, Issue 2, 15 October 2006, Pages 232–239
نویسندگان
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