Nanobody cDNA Mock-Up in pHEN6c Plasmid Vector: Live Out

Whenever there is no adequate DNA replication in vitro, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, E. coli, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured E. coli sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from E. coli by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.

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

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 E. coli 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 −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.