کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2020425 | 1542348 | 2014 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression and purification of recombinant Saccharomyces cerevisiae mitochondrial carrier protein YGR257Cp (Mtm1p)
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
The Saccharomyces cerevisiae mitochondrial carrier YGR257Cp (Mtm1p) is an integral membrane protein that plays an essential role in mitochondrial iron homeostasis and respiratory functions, but its carrier substrate has not previously been identified. Large amounts of pure protein are required for biochemical characterization, including substrate screening. Functional complementation of a Saccharomyces knockout by expression of TwinStrep tagged YGR257Cp demonstrates that an affinity tag does not interfere with protein function, but the expression level is very low. Heterologous expression in Pichia pastoris improves the yield but the product is heterogeneous. Expression has been screened in several Escherichia coli hosts, optimizing yield by modifying induction conditions and supplementing with rare tRNAs to overcome codon bias in the eukaryotic gene. Detection of an additional N-terminal truncation product in E. coli reveals the presence of a secondary intracistronic translation initiation site, which can be eliminated by silent mutagenesis of an alternative (Leu) initiation codon, resulting in production of a single, full-length polypeptide (â¼30% of the total protein) as insoluble inclusion bodies. Purified inclusion bodies were successfully refolded and affinity purified, yielding approximately 40 mg of pure, soluble product per liter of culture. Refolded YGR257Cp binds pyridoxal 5â²-phosphate tightly (KD < 1 μM), supporting a new hypothesis that the mitochondrial carrier YGR237Cp and its homologs function as high affinity PLP transporters in mitochondria, providing the first evidence for this essential transport function in eukaryotes.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 93, January 2014, Pages 77-86
Journal: Protein Expression and Purification - Volume 93, January 2014, Pages 77-86
نویسندگان
Mei M. Whittaker, James W. Whittaker,