کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
6489751 1416541 2018 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Physicochemical improvement of rabbit derived single-domain antibodies by substitutions with amino acids conserved in camelid antibodies
ترجمه فارسی عنوان
بهبود فیزیکوشیمیایی آنتی بادی های تک دامنه حاصل از خرگوش ها با جایگزینی با اسید های آمینه حفاظت شده در آنتی بادی های شترید
کلمات کلیدی
آنتیبادی تک دامنه، منطقه متغیر زنجیره سنگین خرگوش، بهبود فیزیکوشیمیایی، جایگزینی اسید آمینه، پایداری حرارتی، اتصال غیر اختصاصی، ثبات کلوئیدی،
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
چکیده انگلیسی
Recently, we showed that immunized rabbit heavy chain variable regions (rVHs) can have strong antigen binding activity comparable to that of the camelid variable domain of the heavy chain of heavy chain antibody (VHH). These rVHs lack the light chain variable regions (rVLs), which exist in the authentic Fab format; thus, molecular surfaces at the interface region of rVHs are exposed to solvent. This physical feature may change physicochemical properties, such as causing reduced stability. By overcoming potential physicochemical issues through engineering the interface region, rVHs could become more useful as single-domain antibodies. In this study, we substituted amino acid residues conserved at the interface region of rVHs with those of VHHs. These substitutions included V37F, involving substitution of a residue in the hydrophobic core with a bulkier hydrophobic amino acid, and G44E/L45R, involving double substitutions of highly exposed residues with more hydrophilic ones. As expected, biophysical and structural characterizations showed that the V37F substitution markedly enhanced the thermal stability through increased hydrophobic packing, while G44E/L45R substitutions greatly reduced hydrophobicity of the interface. The quadruple substitutions of V37F/G44E/L45R/F91Y resulted in not only enhancements of thermal stability and reduction in hydrophobicity, both in an additive manner, but also synergistic improvement of purification yield. This quadruple mutant exhibited greatly reduced non-specific binding with improved colloidal stability owing to the reduced hydrophobicity. The approach used in this study should further enhance the utility of rVHs and promote research and development of single-domain antibodies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Bioscience and Bioengineering - Volume 125, Issue 6, June 2018, Pages 654-661
نویسندگان
, , , ,