A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae.

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.

Glutathione kinetically outcompetes reactions between dimedone and a cyclic sulfenamide or physiological sulfenic acids.

Dimedone and its derivates are used as selective probes for the nucleophilic detection of sulfenic acids in biological samples. Qualitative analyses suggested that dimedone also reacts with cyclic sulfenamides. Furthermore, under physiological conditions, dimedone must compete with the highly concentrated nucleophile glutathione. We therefore quantified the reaction kinetics for a cyclic sulfenamide model peptide and the sulfenic acids of glutathione and a model peroxiredoxin in the presence or absence of dimedone and glutathione. We show that the cyclic sulfenamide is stabilized at lower pH and that it reacts with dimedone. While reactions between dimedone and sulfenic acids or the cyclic sulfenamide have similar rate constants, glutathione kinetically outcompetes dimedone as a nucleophile by several orders of magnitude. Our comparative in vitro and intracellular analyses challenge the selectivity of dimedone. Consequently, the dimedone labeling of cysteinyl residues inside living cells points towards unidentified reaction pathways or unknown, kinetically competitive redox species.

Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum.

Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of PfKelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of PfKelch13 resulted in ring-stage survival rates around 40%. While the loss of the thiol group of C469 or of the potential disulfide bond between residues C580 and C532 had no effect on the artemisinin susceptibility, the thiol group of C473 could not be replaced. Furthermore, we detected two different forms of PfKelch13 with distinct electrophoretic mobilities around 85 and 95 kDa, suggesting an unidentified post-translational modification. We also established a protocol for the production of recombinant PfKelch13 and produced an antibody against the protein. Recombinant PfKelch13 adopted alternative oligomeric states and only two of its seven cysteine residues, C469 and C473, reacted with Ellman's reagent. While common field mutations resulted in misfolded and completely insoluble recombinant PfKelch13, cysteine-to-serine replacements had no effect on the solubility except for residue C473. In summary, in contrast to residues C469, C532, and C580, the surface-exposed thiol group of residue C473 appears to be essential. However, not the redox properties but impaired folding of PfKelch13, resulting in a decreased PfKelch13 abundance, alters the artemisinin susceptibility and is the central parameter for mutant selection.