The rapid emergence of antibiotic resistant bacteria is a pressing concern, threatening public health and food security worldwide. Antibiotic resistance commonly originates through conjugative plasmid transfer (CPT), where plasmids are transferred and replicated between bacteria. Although the overall mechanism of CPT is known, many plasmid-encoded proteins essential for CPT remain unstudied. One such unstudied plasmid encoded protein is orf90, a putative transcriptional regulator located on the multiresistant plasmid pSK41 from Staphylococcus aureus. Methicillin resistant S. aureus (MRSA) is considered a serious threat by the Centers for Disease Control and Prevention (CDC), and how it acquires resistance plasmids is still an open area of study. In this investigation, we sought to gain valuable insight into the structure and character of orf90. We successfully expressed and purified two orf90 constructs, an untagged version (His-orf90) and a maltose binding protein tagged version (His-MBP-orf90). We hypothesize that orf90 will bind the pSK41 origin of transfer and that one of our constructs will yield usable crystals for structure solution. Crystallography trials for His-Orf90 did not yield macromolecular crystals, possibly due to the sample purity. Binding of His-Orf90 to a 205 bp region of pSK41 containing the conserved origin of transfer (oriT) was confirmed via EMSA (Electrophoretic Mobility Shift Assay). Additionally, crystallization screens with His-MBP-orf90 identified three favorable conditions for crystallization. We have shown that orf90 binds to the pSK41 oriT and that His-MBP-orf90 is a promising crystallization construct. Characterizing pSK41 Orf90’s structure and function will further enhance our understanding of CPT, potentially yielding useful data for research aimed at combating antibiotic resistance.
