A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae
Authors:Katrin Hieronimus1,2,#, Tabea Donauer1,2,#, Jonas Klein1,#, Bastian Hinkel1,#, Julia Vanessa Spänle1,#, Anna Probst1,#, Justus Niemeyer1,#, Salina Kibrom1, Anna Maria Kiefer1, Luzia Schneider2, Britta Husemann2, Eileen Bischoff2, Sophie Möhring2, Nicolas Bayer1, Dorothée Klein1, Adrian Engels1, Benjamin Gustav Ziehmer2, Julian Stieß3, Pavlo Moroka1, Michael Schroda1, and Marcel Deponte2
doi: 10.15698/mic2024.04.821
Volume 11, pp. 128 to 142, published 30/04/2024.
1 Faculty of Biology, Molecular Biotechnology & Systems Biology, RPTU Kaiserslautern, D-67663 Kaiserslautern, Germany.
2 Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663 Kaiserslautern, Germany.
3 Faculty of Computer Science, RPTU Kaiserslautern, D-67663 Kaiserslautern, Germany.
# Authors contributed equally to this work.
Keywords:
iGEM, Leishmania tarentolae, LEXSY expression, MoClo, recombinant protein production, SARS-CoV-2, spike protein.
Corresponding Author(s):
Conflict of interest statement:
The authors declare that there are no conflicts of interest.
Please cite this article as:
Katrin Hieronimus, Tabea Donauer, Jonas Klein, Bastian Hin-kel, Julia Vanessa Spänle, Anna Probst, Justus Niemeyer, Sa-lina Kibrom, Anna Maria Kiefer, Luzia Schneider, Britta Huse-mann, Eileen Bischoff, Sophie Möhring, Nicolas Bayer, Dorothée Klein, Adrian Engels, Benjamin Gustav Ziehmer, Julian Stieß, Pavlo Moroka, Michael Schroda, and Marcel Deponte (2024). A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae. Microbial Cell 11: 128-142. doi: 10.15698/mic2024.04.821
© 2024 Hieronimus et al. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.
Abstract:
Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.