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.
References
[1]
Muyldermans, S. (2013) Nanobodies: Natural Single-Domain Antibodies. Annual Review of Biochemistry, 82, 775-797.
https://doi.org/10.1146/annurev-biochem-063011-092449
[2]
Baneyx, F. (1999) Recombinant Protein Expression in Escherichia coli. Current Opinion in Biotechnology, 10, 411-421. https://doi.org/10.1016/S0958-1669(99)00003-8
[3]
Stoyanov, J.V. and Brown, N.L. (2003) The Escherichia coli Copper-Responsivecopa Promoter Is Activated by Gold. Journal of Biological Chemistry, 278, 1407-1410. https://doi.org/10.1074/jbc.C200580200
[4]
Conrath, K.E., Lauwereys, M., Galleni, M., Matagne, A., Frere, J.-M., Kinne, J., Wyns, L. and Muyldermans, S. (2001) Beta-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the Camelidae. Antimicrobial Agents and Chemotherapy, 45, 2807-2812.
https://doi.org/10.1128/AAC.45.10.2807-2812.2001
[5]
Baral, T.N., Magez, S., Stijlemans, B., Conrath, K., Vanhollebeke, B., Pays, E., Muyldermans, S. and De Baetselier, P. (2006) Experimental Therapy of African Trypanosomiasis with a Nanobody-Conjugated Human Trypanolytic Factor. Nature Medicine, 12, 580-584. https://doi.org/10.1038/nm1395
[6]
Abderrazek, R.B., Hmila, I., Vincke, C., Benlasfar, Z., Pellis, M., Dabbek, H., Saerens, D., El Ayeb, M., Muyldermans, S. and Bouhaouala-Zahar, B. (2009) Identification of Potent Nanobodies to Neutralize the Most Poisonous Polypeptide from Scorpion Venom. Biochemical Journal, 424, 263-272.
https://doi.org/10.1042/BJ20090697
[7]
Gu, J., Stephenson, C.G. and Iadarola, M.J. (1994) Recombinant Proteins Attached to a Nickel-NTA Column: Use in Affinity Purification of Antibodies. BioTechniques, 17, 257-261.
[8]
Ghahroudi, M.A., Desmyter, A., Wyns, L., Hamers, R. and Muyldermans, S. (1997) Selection and Identification of Single Domain Antibody Fragments from Camel Heavy-Chain Antibodies. FEBS Letters, 414, 521-526.
https://doi.org/10.1016/S0014-5793(97)01062-4
[9]
Muyldermans, S., Baral, T.N., Retamozzo, V.C., De Baetselier, P., De Genst, E., Kinne, J., Leonhardt, H., Magez, S., Nguyen, V.K., Revets, H., Rothbauer, U., Stijlemans, B., Tillib, S., Wernery, U., Wyns, L., Hassanzadeh-Ghassabeh, G. and Saerens, D. (2009) Camelidimmunoglobulins and Nanobody Technology. Veterinary Immunology and Immunopathology, 128, 178-183.
https://doi.org/10.1016/j.vetimm.2008.10.299
[10]
Shapiro, A.L., Vinuela, E. and Maizel Jr., J.V. (1967) Molecular Weight Estimation of Polypeptide Chains by Electrophoresis in SDS-Polyacrylamide Gels. Biochemical and Biophysical Research Communications, 28, 815-820.
https://doi.org/10.1016/0006-291X(67)90391-9
[11]
Chial, H.J., Thompson, H.B. and Splittgerber, A.G. (1993) A Spectral Study of the Charge Forms of Coomassie Blue G. Analytical Biochemistry, 209, 258-266.
https://doi.org/10.1006/abio.1993.1117
[12]
Compton, S.J. and Jones, C.G. (1985) Mechanism of Dye Response and Interference in the Bradford Protein Assay. Analytical Biochemistry, 151, 369-374.
https://doi.org/10.1016/0003-2697(85)90190-3
[13]
Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. Proceedings of the National Academy of Sciences of the United States of America, 76, 4350-4354. https://doi.org/10.1073/pnas.76.9.4350
[14]
Primorose, S.B. and Twyman, R.M. (2006) Principles of Gene Manipulation and Genomics. 7th Edition. Blackwell Publishing, Malden.
[15]
Saerens, D. (2010) Isolation and Optimization of Camelid Single-Domain Antibodies: Dirk Saerens’ Work on Nanobodies. World Journal of Biological Chemistry, 1, 235-238. https://doi.org/10.4331/wjbc.v1.i7.235
[16]
Baneyx, F. and Mujacic, M. (2004) Recombinant Protein Folding and Misfolding in Escherichia coli. Nature Biotechnology, 22, 1399-1408.
https://doi.org/10.1038/nbt1029
[17]
Bernkop-Schnürch, A. and Scerbe-Saiko, A. (1998) Synthesis and in Vitro Evaluation of Chitosan-EDTA-Protease-Inhibitor Conjugates Which Might Be Useful in Oral Delivery of Peptides and Proteins. Pharmaceutical Research, 15, 263-269.
https://doi.org/10.1023/A:1011970703087
