Rational Design of the Soluble Variant of <scp>l</scp>-Pipecolic Acid Hydroxylase using the α-Helix Rule and the Hydropathy Contradiction Rule
Suguru Shinoda, Aoi Itakura, Haruka Sasano, Ryoma Miyake, Hiroshi Kawabata, Yasuhisa Asano
Abstract
gene by site-directed mutagenesis. Moreover, the I28P/L142R and C76Y/L142R double variants displayed improved soluble expression levels compared to the single variants. These variants were also more thermostable than the wild-type XdPH. To analyze the effect of the alteration on one of the hotspots, L142 was replaced with various hydrophilic and positively charged residues. The remarkable increase in soluble protein expression caused by the alterations suggests that the decrease in the hydrophobicity of the protein surface and the enhancement of the interaction between nearby residues are important factors determining the solubility of the protein. Overall, this study demonstrated the effectiveness of our protocol in identifying aggregation hotspots for recombinant protein production and in basic biochemical research.