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Digital Publisher

Digital Commons at St. Mary's University

Publication Date

Spring 2025

Keywords

Mycoplasma pneumoniae; CARDS toxin; Community Acquired Respiratory Distress Syndrome;

Description

Mycoplasma pneumoniae is an atypical bacterium that is known to cause respiratory distress and can lead to respiratory illnesses such as community-acquired pneumonia, pharyngitis, and tracheobronchitis. M.pneumoniae produces an ADPribosylation and vacuolating toxin called Community Acquired Respiratory Distress Syndrome (CARDS) toxin. Alone, the CARDS toxin is capable of reproducing many of the signs of an M.pneumoniae infection. The CARDS toxin often causes what is commonly referred to as “walking pneumonia”, a term used to characterize the mild symptoms associated with the infection. Patients with “walking pneumonia” can manage the condition without hospitalization, and often can continue with their normal day-to-day life. The full-length CARDS toxin, which is composed of Domains 1, 2, and 3, includes the amino acids 1 through 591. Domains 2 and 3 include amino acids 269 through 591. When the CARDS toxin reaches the endoplasmic reticulum, domain 1, the mART domain, separates from domains 2 and 3. The mART domain takes part in ADP-ribosylation, which contributes to the pathogenic effects of M. pneumoniae. Currently, researchers are investigating the functions of domains 2 and 3 after their separation from domain 1. To develop a better understanding of domains 2 and 3, it is imperative to purify the portion of the protein responsible for these domains. The mutant CARDS toxin, which includes domains 2 and 3, consists of amino acids 273 through 591. The 273 mutant CARDS toxin, which contains more basic amino acids, has a higher isoelectric point than the full-length CARDS, which contains more acidic amino acids. Understanding that the mutant CARDS toxin has a higher isoelectric point than the full-length toxin, aided in the pH selection for purification. Proteins typically have the least solubility near their isoelectric point. Therefore, during protein purification, selecting a pH higher than the protein’s isoelectric point can minimize protein precipitation, and can improve the purity and yield. The objective of this project was to optimize the protein purification of the mutant CARDS toxin by modifying the current protein purification protocol, particularly concerning the pH of the elution and binding buffers. To familiarize ourselves with protein purification, we began by purifying the full-length CARDS toxin on the AKTA Start HPLC equipped with a HisTrap. We then used column chromatography to determine the optimal pH for the mutant CARDS toxin. After evaluating which pH resulted in the highest protein concentration, we used the AKTA with the corresponding binding buffer and elution buffer for optimal purification. Through column chromatography, we determined, via Coomassie SDS-PAGE gel, that pH 11 elution buffer and binding buffer provided the highest yield of the 273 CARDS protein.

Format

pdf

Size

1 page

City

San Antonio, Texas

Optimized Protein Purification of the Mutant 273 CARDS Toxin from Mycoplasma pneumoniae

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