Recombinant expression and purification of Porphyromonas gingivalis Tyrosine Kinase 1 in Escherichia coli

The goal of this study was to optimize the expression and purification of Porphyromonas gingivalis Tyrosine Kinase 1 (Ptk1) in recombinant Escherichia coli containing the plasmids Gro_ESL and Ptk1, with a His6 tag, to obtain a highly pure protein sample. Various experimental conditions were tested to determine the most effective parameters for protein expression and purification, including adjustments in induction timing, buffer composition, and purification techniques. Optimal protein expression was achieved when cells were induced at an optical density (OD) of 0.3 to 0.4. β-D-1-thiogalactopyranoside (IPTG), which activates the lac operon, was used to induce Ptk1 expression. A concentration of both 0.25 and 0.5 mM IPTG were found to be sufficient for maximizing expression while minimizing unintended protein production. Purification was carried out using immobilized metal ion affinity chromatography (MIAC), which selectively binds the His6-tagged Ptk1. Wash buffers containing imidazole at concentrations between 20 and 35 mM were tested to improve purification efficiency. While these trials resulted in a significantly purer product, SDS-PAGE electrophoresis with Coomassie staining identified two to three contaminant proteins persisting across all trials. This study made significant progress in identifying optimal techniques for future work, despite some possible impurities in the product. Further testing is needed to quantify the purity of the product. This study provides a strong foundation for refining protein purification strategies and improving yield, ultimately advancing the study of Ptk1 function and its role in P. gingivalis pathogenesis.