کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8359323 | 1542290 | 2018 | 27 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Evaluation of in vitro refolding vs cold shock expression: Production of a low yielding single chain variable fragment
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کلمات کلیدی
GSHMRCGSSGIMACTBSGdnHClCAIscFvIPTGDMSO - DMSOE.coli - اشریشیا کلیEscherichia coli - اشریشیا کُلیisopropyl β-D-1-thiogalactopyranoside - ایزوپروپیل β-D-1-thiogalactopyranosideTris buffered saline - تریس نمک بافرDimethyl sulfoxide - دیمتیل سولفواکسیدprothrombin time - زمان پروترومبینCodon adaptation index - شاخص سازگاری کدونFusion partner - شریک فیوژنMedical Research Council - شورای تحقیقات پزشکیCold shock - شوک سردTissue factor - عامل بافتSingle chain variable fragment - قطعه متغیر زنجیره ای تنهاInclusion bodies - مجازات شاملDisulfide bridge - پل دی سولفیدreduced glutathione - کاهش گلوتاتیونimmobilized metal affinity chromatography - کروماتوگرافی وابسته به فلز متمرکزoxidized glutathione - گلوتاتیون اکسید شده
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The development of therapeutic antibodies in their various forms has been a constant challenge since the development of the first monoclonal antibodies in 1975. This is especially true for the development of therapeutic single chain variable (scFv) fragments in Escherichia coli. In a previous study the selection of a tissue factor inhibiting single chain variable fragment (TFI-scFv) isolated from the Thomlinson IÂ +Â J phage libraries was described. Although the initial findings were promising, additional characterization of the antibody fragment and subsequent application was hampered due low protein yield. This study reports on: i) the improved expression of a previously low yielding TFI-scFv in the cytoplasm of E. coli BL21 (DE3) through modifications to the expression systems in conjunction with codon optimization ii) evaluation of two commercial methods of protein recovery: in vitro refolding and the utilization of cold shock expression systems in conjunction with E. coli SHuffle. Results showed that TFI-scFv could be expressed at higher levels in the cytoplasm of E. coli than previously achieved in the periplasm. Both the in vitro refolding and cold shock strategies were capable of producing functional TFI-scFv with varying degrees of success. These procedures could be applied to improve the production of other problematic low yielding scFv isolated from phage display repositories in order to facilitate their characterization.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 151, November 2018, Pages 62-71
Journal: Protein Expression and Purification - Volume 151, November 2018, Pages 62-71
نویسندگان
Jan-G. Vermeulen, Felicity Burt, Esta van Heerden, Errol Cason, Muriel Meiring,