کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10880610 | 1076986 | 2005 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Molecular engineering of an EGFP/disintegrin-based integrin marker
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موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوشیمی، ژنتیک و زیست شناسی مولکولی (عمومی)
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چکیده انگلیسی
Disintegrins are viper venom peptides, which bind integrins with high affinity (10â8 M) and selectivity. Among them, eristostatin (Er) selectively binds and inhibits αIIbβ3 integrin function. In this work we have engineered an enhanced green fluorescence protein (EGFP)-tagged Er as an αIIbβ3 biomarker to be used in bioassays involving fluorescence detectors. For this, we have first constructed an EGFP bacterial expression vector, which resulted in a 6xHis tag-coding region followed by the EGFP gene and a 3â² multiple cloning site (MCS) comprising nine restriction sites. This vector, termed pAZ, was used to clone the Er gene, resulting in a 32 kDa EGFP-Er fusion protein when expressed as characterized by SDS-PAGE and Western blot. Both EGFP-Er and EGFP (expressed from the empty pAZ vector) were purified by immobilized metal affinity chromatography (IMAC) and their fluorescence was measured showing similar values, thus suggesting that the Er portion is not affecting the EGFP activity. EGFP-Er, but not EGFP selectively bound to immobilized platelets as detected by confocal microscopy indicating the preservation of Er disintegrin activity and its potential use as a marker for αIIbβ3 integrin. Our data suggest the use of the pAZ vector for expressing soluble EGFP-labeled proteins and the use of EGFP-fused disintegrins as markers for integrins.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Toxicon - Volume 46, Issue 2, August 2005, Pages 178-184
Journal: Toxicon - Volume 46, Issue 2, August 2005, Pages 178-184
نویسندگان
Diego Butera, Roxane Maria Fontes Piazza, Mary Ann McLane, Roger Chammas, Ana Maria Moura da Silva,