Molecular Cloning, Expression and Purification of Recombinant VHH Proteins Expressed in <i>E. coli</i>  

作　　者：Tewodros Fentahun Jember Tewodros Fentahun Jember(Department of Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia)

机构地区：[1]Department of Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia

出　　处：《American Journal of Molecular Biology》2021年第4期129-141,共13页美国分子生物学期刊（英文）

摘　　要：Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in <em>E. coli</em> WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD₂₈₀ nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and <span style="white-space:nowrap;">&minus;</span>0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in <em>E. coli</em> WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD₂₈₀ nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and <span style="white-space:nowrap;">&minus;</span>0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.

关 键 词：VHH NANOBODY Protein 

分 类 号：R73[医药卫生—肿瘤]