Differences in the purification and solution properties of PurC gene products from Streptococcus pneumoniae and Bacillus anthracis
Section snippets
1. Introduction
The exponentially rising threat of antimicrobial resistance among deadly pathogens has garnered the focus of the scientific community in recent years. Among these pathogens are the Gram-positive bacteria Streptococcus pneumoniae and Bacillus anthracis. Both are associated with a decrease in the potency of available treatments [1], [2], [3], [4], [5], and at the forefront of continuing efforts to discover new antibiotics by targeting novel enzymes in essential metabolic pathways [6], [7]. One
2.1. Chemicals and reagents
All chemicals for buffers, media and solutions were purchased from Thermo Fisher Scientific, unless explicitly stated.
2.2. Cloning of PurC constructs
PurC genomic DNA from B. anthracis (ΔANR strain; AE017334.2) was inserted into the pET-15b expression vector (Novagen) to create a fusion protein construct with an added N-terminal sequence of MGSSHHHHHHSSGLVPRGSH to include a His-tag and a thrombin cleavage site. The vector was transformed into 5′ϒ Z-competent Escherichia coli cells (Zymo Research) for DNA isolation and
3.1. Molecular masses
The calculated masses, including the added N-terminal segment, was 29,700.8 Da for BaPurC and 29,166.0 Da for SpPurC. Mass spectroscopy analysis resulted in a mass of 29,569.3 Da for BaPurC, a difference of 131.5 Da, and 29,034.9 Da for SpPurC, a difference of 131.1 Da. Thus, the initial methionine, prior to the histidine tag, was absent. Otherwise, each protein (BaPurC and SpPurC) was as designed.
3.2. Protein yield
The whole cell electrophoresis gel intensities of carefully controlled, paired growth of SpPurC and Ba
4. Discussion
There have been several crystal structures of PurCs solved [20], [24], [25] and its reaction mechanism proposed [20], but to our knowledge no high throughput data are available. This may be due to difficulties in obtaining large quantities of viable protein to perform high throughput screening to identify new antimicrobials targeting the de novo purine pathway of pathogens. Our data showed that there are clear differences in the solubility properties between BaPurC and SpPurC, despite their
5. Conclusion
PurC is an excellent drug targeting protein. To identify inhibitors, large amounts of recombinant proteins are needed. Two PurC proteins, from Gram-positive S. pneumoniae and B. anthracis exhibit high sequence similarity and identity. However, as we have shown here, they exhibit very different solution properties, leading to a ten-fold difference in yield in their recombinant protein preparation, with about 50 mG per gram of cells for His-tagged SpPurC, but only 5 mG per gram of cells for
Conflict of interest
The authors declare that there are no conflicts of interest.
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Acknowledgments
We would like to thank Dr. Shahila Mehboob and Claire Marquis from the Center for Pharmaceutical Biotechnology at the University of Illinois at Chicago, for PurC plasmid construction. Also Dr. Mehboob provided the initial activity assay conditions. We also thank a reviewer suggesting binding studies to hydrophobic surfaces.
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