Rational Design and Directed Evolution of Human Paraoxonase I (huPON1) for Increased Solubility and Stability in E. coli

Paraoxonase I (PON1) is a serum hydrolase that has been found to hydrolyze organophosphates (OP), such as VX and sarin. Because human PON1 (huPON1) is a human protein with reasonable activity, it seems like a promising therapeutic agent against OP poisoning. However, huPON1 is an insoluble protein and not very stable when expressed in E. coli, whereas a chimeric mammalian PON1 variant, G2E6, has proven to be soluble and expressible in E. coli. We are specifically working on a way to make huPON1 more soluble by using a rational approach of changing a cluster of hydrophobic residues on the surface of the protein into polar ones. Since these mutations are on the surface of the protein, it is rationalized that it may not significantly affect the activity of huPON1. We are employing two different rational designs to make huPON1 more soluble. The first strategy utilizes a three fragment PCR overlap containing 12 hydrophobic residues from the HDL binding site that are mutated to polar residues. The second design utilizes total gene synthesis in order to make 16 hydrophobic to polar residues in huPON1 corresponding to the G2E6 mutations that are polar. These variants were put into a vector containing folding reporter GFP to screen for solubility and expression by means of cellular fluorescence. We have been able to express both variants and have observed them to be significantly more soluble than the wild-type one. In order to achieve even more solubility, we have fused these huPON1 variants with Maltose Binding Protein (MBP) which has been shown to improve the solubility of many proteins. By doing this, we have been able to express and purify large amounts of protein in order to obtain biophysical characteristics. Both huPON1 variants prove to be active against paraoxon and phenyl acetate.NIH U54 NS058183 "Center for Catalytic Bioscavenger Medical Defense Research"A two-year embargo was granted for this item