Development of design method to get soluble protein

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In order to know structure and function of gigantic protein and protein complex, a method to cut and take out stable partial structure of protein and a method to make the partial structure soluble are needed. Our purpose is to develop a design method to take out partial structure of protein in a soluble and stable from. We intended further to verify that the designed partial structure forms soluble and stable structure in solvent. In process of molecular evolution, a fusion of domain or module occurred frequently. We showed that in reverse transcriptase, on the occasion of RNaseH domain fusion, at least 4 amino acid replacements occurred in adopting to the atomic interactions at domain contact. We applied the same method to make a partial protein structure soluble. Previously we found a barnase-like domain in RNA polymerase. We planed to cut out the barnase-like domain from RNA polymerase and designed a soluble form of isolated barnase-like domain. Then we did a chemical synthesis of the sequence. For getting soluble barnase-like domain, we established a method to replace amino acid residues using an evolutionary replacement pattern of amino acid in domain fusion. This method is applicable for the proteins whose three-dimensional structures are unknown. Also, we designed a mini-barnase by removing a module from barnasr, performed molecular dynamics and evaluated its solubility and stability by free energy calculation. Mini-barnase lacking 26 amino acid residues was chemically synthesized. We measured CD and NMR of designed mini-barnase. It was dissolved into water and took a stable structure similar to the conformation of a barnase except the excised region. Through this research, we developed a method that is useful in cutting and taking out a part of protein in soluble and stable form.