کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6488057 | 1416354 | 2018 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma](/preview/png/6488057.png)
چکیده انگلیسی
The Escherichia coli phosphoglucose isomerase (pgi) mutant strain GALG20 was developed previously from wild-type K12 strain MG1655 for increased plasmid yield. To investigate the potential effects of the pgi deletion/higher plasmid levels on recombinant human Interferon Gamma (IFN-γ) production, a detailed network of the central metabolic pathway (100 metabolites, 114 reactions) of GALG20 and MG1655 was constructed. Elementary mode analysis (EMA) was then performed to compare the phenotypic spaces of both the strains and to check the effect of the pgi deletion on flux efficiency of each metabolic reaction. The results suggested that pgi deletion increases amino acid biosynthesis and flux efficiency towards IFN-γ synthesis by 11%. To further confirm the qualitative prediction that the pgi mutation favours recombinant human IFN-γâ¯expression, GALG20 and MG1655 were lysogenised, transformed with a plasmid coding for IFN-γ and tested alongside with BL21(DE3) for their expression capabilities in shake flask experiments using complex media. IFN-γ gene expression was analysed by quantifying plasmid and mRNA copy number per cell and IFN-γâ¯protein production level. Specific IFN-γ yields confirmed the in silico metabolic network predictions, with GALG20(DE3) producing 3.0-fold and 1.5-fold more IFN-γ as compared to MG1655(DE3) and BL21(DE3), respectively. Most of the total IFN-γ was expressed as inclusion bodies across the three strains: 95% in GALG20(DE3), 97% in BL21(DE3) and 72% in MG1655(DE3). The copy number of mRNA coding for IFN-γ was found to be higher in GALG20(DE3) as compared to the other two strains. Overall, these findings show that GALG20(DE3) has the potential to become an excellent protein expression strain.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Enzyme and Microbial Technology - Volume 117, October 2018, Pages 23-31
Journal: Enzyme and Microbial Technology - Volume 117, October 2018, Pages 23-31
نویسندگان
Rajat Pandey, Nitin Kumar, Gabriel A. Monteiro, Venkata Dasu Veeranki, D.M.F. Prazeres,