Version 1
: Received: 7 April 2024 / Approved: 7 April 2024 / Online: 8 April 2024 (10:34:10 CEST)
How to cite:
Torres, S.D.C.; Castro-Amarante, M.F.; Ferreira, L.C.S.; Guskov, A.; Balan, A. Successful production of antibodies against extra cytoplasmic loops of the Mycobacterium tuberculosis ABC transporter Rv1819c. Preprints2024, 2024040481. https://doi.org/10.20944/preprints202404.0481.v1
Torres, S.D.C.; Castro-Amarante, M.F.; Ferreira, L.C.S.; Guskov, A.; Balan, A. Successful production of antibodies against extra cytoplasmic loops of the Mycobacterium tuberculosis ABC transporter Rv1819c. Preprints 2024, 2024040481. https://doi.org/10.20944/preprints202404.0481.v1
Torres, S.D.C.; Castro-Amarante, M.F.; Ferreira, L.C.S.; Guskov, A.; Balan, A. Successful production of antibodies against extra cytoplasmic loops of the Mycobacterium tuberculosis ABC transporter Rv1819c. Preprints2024, 2024040481. https://doi.org/10.20944/preprints202404.0481.v1
APA Style
Torres, S.D.C., Castro-Amarante, M.F., Ferreira, L.C.S., Guskov, A., & Balan, A. (2024). Successful production of antibodies against extra cytoplasmic loops of the Mycobacterium tuberculosis ABC transporter Rv1819c. Preprints. https://doi.org/10.20944/preprints202404.0481.v1
Chicago/Turabian Style
Torres, S.D.C., Albert Guskov and Andrea Balan. 2024 "Successful production of antibodies against extra cytoplasmic loops of the Mycobacterium tuberculosis ABC transporter Rv1819c" Preprints. https://doi.org/10.20944/preprints202404.0481.v1
Abstract
Antibodies are important tools for the study of membrane proteins in biological, biophysics and structural analyses. However, the production of purified membrane proteins is still a formidable challenge due to the amphipathic character and inherent instability of these proteins. In this work, we tested a strategy for producing antibodies against an ATP-Binding Cassette (ABC) transporter without the need to produce the full membrane complex. Instead, just the extra cytoplasmic regions of the ABC-type transporter Rv1819c from Mycobacterium tuberculosis were presented in the RAD-display, a scaffold system based on the engineered Pyrococcus furiosus RadA protein developed for exposition of peptides. The RadA scaffold protein is highly stable, can tolerate long insertions and easy to produce in Escherichia coli. Based on the three-dimensional structure of Rv1891c, we selected two versions of the main extracellular loops of the permease to use as test cases for this approach. Rv1819c-derived peptides displayed on RAD display system were used for immunization of mice and production of polyclonal antibodies. These antibodies were able to recognize the transporter in M. tuberculosis cell extracts, on intact cells by flow cytometry and as purified protein. The results showed the approach can be a useful tool for in vitro analyses, screening, and evaluation of different epitopes in membrane proteins.
Keywords
ABC transporters; Mycobacterium tuberculosis; antibody-mediated recognition; membrane proteins; RAD display
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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