Bacterium Listeria monocytogenes is an intracellular pathogen with the ability to spread between cells and divide in the cytosol of host cells. During intracellular cycle it becomes entrapped inside the vacuole, from which it escapes using virulence factors, including metalloprotease Mpl. Due to lack of information for this protein, we decided to isolate and characterize it. We prepared constructs which differed in affinity tag, signal peptide, sequence – either pro or mature form of Mpl. Cultivation of bacterium and expression of Mpl under initial conditions, showed that proMpl is expressed but remains in insoluble fraction, but mature Mpl is not even expressed. The goal became to obtain a soluble form of proMpl. We approached this challenge systematically, changing or introducing one or two variables at a time. Firstly, we checked the influence of i) temperature and time of expression, ii) lysis buffer, iii) bacterial lysis method, but none of the listed variables increased protein solubility. Results from NaDS-PAGE showed the presence of an unknow protein the size of approximately 40 kDa, which is most probably a bacterial protein. We continued the work by preparing a construct that allows expression of proMpl in the periplasmic space of the bacteria but did not see an increase in the solubility. The next assumption was that when proMpl is overexpressed it has a higher tendency to aggregate. We tested the influence of inducer concentration and type of bacterial medium, which was also not a solution. Next, we assumed that proMpl is not able to reach its native conformation, thus we prepared a construct which enables expression of proMpl without additional amino acid residues. We expressed proMpl from this newly prepared construct in the presence and absence of external zinc added to the bacterial culture medium. proMpl contains one zinc atom in its active site, therefore it could have an impact on correct folding. We also tried to isolate proMpl from the periplasmic space using modified osmotic shock, but this method was not the solution. We show that the expression of recombinant proMpl requires special conditions, which we did not discover despite a systematic search. Lastly, we determined the model of three-dimensional structure using AlphaFold2. The model predicts some unstructured regions, and we can also observe the importance of the propeptide. We assume that by systematically preparing various proMpl constructs based on AlphaFold2 model, we could prepare those that would be successfully expressed in E. coli.
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