کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020941 1069218 2010 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Construction, expression and characterization of a chimeric multi-domain protein mediating specific DNA transfer
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Construction, expression and characterization of a chimeric multi-domain protein mediating specific DNA transfer
چکیده انگلیسی

The delivery of plasmid DNA to target cells using a simple, defined, non-viral system is an area of intense research in gene therapy. Here, we describe a novel DNA carrier protein termed TG, consisting of the DNA-binding domain of the yeast transcriptional activator GAL4 and human immunodeficiency virus type 1 Tat protein, which can transfer modified naked plasmid DNA into target cells to express foreign genes of interest. The TG protein was expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS–PAGE and Western blotting revealed that the fusion protein was highly expressed with a yield of approximately 275 mg/L. We also constructed the pIRES-UAS-EGFP DNA vector, consisting of upstream activating sequences (UASs) for the specific binding of the DNA-binding protein and the enhanced green fluorescent protein (EGFP) gene. The TG protein could bind specifically to pIRES-UAS-EGFP, forming a complex which could efficiently transfect target cells and result in detectable EGFP protein expression. Thus, these results provide a basis for development of efficient non-viral DNA transfer vectors for further improvements of gene therapy strategies.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 74, Issue 2, December 2010, Pages 189–195
نویسندگان
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