Use of Small Molecules in the Aid of Solubility of E. coli P3340 during Lysis Stage of Purification

Protein SFBNYU_003340 (P3340) is a surface protein of mice-specific segmented filamentous bacteria (SFB), gut bacteria distinguished for their role in development of Th17 and Th22 immune cells. These two subgroups of immune cells are known for their role in fighting pathogens. Protein P3340 homologous protein, P00774 from human-specific SFB, shares 53.6% similarities with P3340 on amino acid level. Protein P3340 also carries two epitopes that were immunodominant in the stimulation of antigen specific mucosal Th17 cells. Still, there is no known target for P3340 protein in intestinal epithelial cells (IEC) or mucus covering IEC. In order to study the function of P3340, we have expressed and purified this protein from E. coli in a N-terminal double His6-tag fusion and without a signal sequence (first 33 amino acids). Although we have produced the protein in our lab from synthetic DNA that was codon-optimized for E. coli and optimized expression and solubility in several buffers, yields were still very low. In this work, we wanted to test different chemicals generally known to increase the solubility of different proteins. One study has shown that adding of certain chemicals at the lysis stage of purification, when cell walls are broken down to release the soluble protein into the solution, significantly increased the yield of some of tested proteins. Before this publication, various molecules were typically added to already purified proteins to increase their solubility and stability prior to protein concentration and storage. In our study, different chemicals (trehalose, glycine betaine, mannitol, L-arginine, potassium citrate, copper (II)-chloride, proline, xylitol, CTAB, glycerol, and dipotassium phosphate) were separately added into the cell lysates of E. coli BL21(DE3) bacteria producing 2xHis6-P3340. Proteins from each lysate were then purified on Ni-NTA, concentration of protein 2xHis6-P3340 was measured in Bradford assay, and purity checked on SDS-PAGE gels.