کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843947 | 1069297 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification, and characterization of rat protein tyrosine phosphatase η catalytic domain
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
Receptor-like protein tyrosine phosphatases generally contain one or two conserved intracellular catalytic domains with a conserved sequence motif ([I/V]HCXAGXXR[S/T]G), a single transmembrane domain, and an external highly variable part. Here, we describe cloning of the intracellular catalytic domain of the rat protein tyrosine phosphatase η (rPTPηCD) into pET28a(+) vector, its expression in Escherichia coli, purification and initial characterization. The purification of His6-tagged rPTPηCD to near homogeneity was achieved by a combination of affinity and size exclusion chromatography. The His-tag was subsequently removed by thrombin digestion. PhastGel IEF electrophoresis demonstrated that the isoelectric point of this 41 kDa His6-tag free recombinant protein was 7.3, which is just slightly higher than the theoretically predicted value of 7.2. To assess the functionality of the rPTPηCD we used the pNPP hydrolysis assay and observed that the enzyme has a specific activity of 9 nmol/min/μg. The secondary structure and stability of the recombinant protein was also analyzed by circular dichroism and fluorescence spectroscopy. In summary, the rPTPηCD is stable at 18 °C, properly folded, and fully active, which makes it a suitable candidate for structural and functional studies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 41, Issue 1, May 2005, Pages 113-120
Journal: Protein Expression and Purification - Volume 41, Issue 1, May 2005, Pages 113-120
نویسندگان
Maria A.M. Santos, Silvia M. Santos, Huita C. Matozo, Rodrigo V. Portugal, Rodolfo Iuliano, Alfredo Fusco, Igor Polikarpov,