کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9902250 | 1545796 | 2005 | 21 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A universal strategy for stable intracellular antibodies
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کلمات کلیدی
EGFRScFv fragmentso-nitrophenyl β-D-galactopyranosideONPGX-GalECLHRPscFveGFPIPTGMBPPBSmAbMonoclonal antibody - آنتی بادی مونوکلونالIntracellular antibodies - آنتی بادی های داخل سلولیisopropyl β-D-thiogalactopyranoside - ایزوپروپیل β-D-thiogalactopyranosideenhanced chemiluminescence - بهبود شیمیایی لومنEnzyme-linked immunosorbent assay - تست الیزاELISA - تست الیزاIntrabodies - داخل بدنendoplasmic reticulum - شبکه آندوپلاسمی Phosphate-buffered saline - محلول نمک فسفات با خاصیت بافریpolymerase chain reaction - واکنش زنجیره ای پلیمرازPCR - واکنش زنجیرهٔ پلیمرازHorseradish peroxidase - پراکسیداز هوررادیشenhanced green fluorescent protein - پروتئین فلورسنت سبز افزایش یافته استFusion protein - پروتئین فیوژن یا پروتئین همجوشیmaltose-binding protein - پروتئین متصل به مالتوزMolecular chaperone - چاپرون مولکولیEpidermal growth factor receptor - گیرنده فاکتور رشد اپیدرمالsingle-chain variable fragment of an antibody - یک قطعه متغیر تک زنجیره ای از آنتی بادی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
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چکیده انگلیسی
The expression of intracellular antibodies (intrabodies) in mammalian cells has provided a powerful tool to manipulate microbial and cellular signalling pathways in a highly precise manner. However, several technical hurdles have thus far restricted their more widespread use. In particular, single-chain antibodies (scFvs) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a preselection step was applied to identify these rare scFvs that could fold properly in the absence of disulfide bonds. Recently, we reported that scFvs can be efficiently expressed within the cytoplasm of bacteria when fused at the C-terminus of the Escherichia coli maltose-binding protein (MBP). Here, we demonstrate that such MBP-scFvs are similarly stabilized when expressed in the mammalian cell cytoplasm as well as other compartments. This was demonstrated by comparing MBP-scFv fusions to the corresponding unfused scFvs that activate a defective β-galactosidase enzyme, others that neutralize the wild-type β-galactosidase enzyme, and an antibody that blocks the epidermal growth factor receptor. In all cases, the MBP-scFvs significantly outperformed their unfused counterparts. Our results suggest that fusion of scFvs to MBP, and possibly to other “chaperones in the context of a fusion protein,” may provide a universal approach for efficient expression of intrabodies in the mammalian cell cytoplasm. This strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv-phage libraries already in existence to identify intrabodies that will be active in vivo.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 303, Issues 1â2, August 2005, Pages 19-39
Journal: Journal of Immunological Methods - Volume 303, Issues 1â2, August 2005, Pages 19-39
نویسندگان
Shelly Shaki-Loewenstein, Rahely Zfania, Stephen Hyland, Winfried S. Wels, Itai Benhar,