Correct Refolding of Precipitated Enzymes Obtained in Recombinant DNA Process by Means of Induction Devices

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(1) Objective of the present study is to design the process for refolding precipitated protein produced by the recombinant DNA method correctly to its authentic 3D-structure. Another aim is to examine possibility of induced refolding, i.e., to test if there is any substance that enhance refolding.(2) Intact hen egg-white lysozyme(Lyzm) and the fully reduced Lyzm preparation were adopted in the present research. Refolding yield was evaluated by CD measurement as well as by the recovered enzyme activity.(3) Intact Lyzm with four S-S bonds was confirmed to be in the random coil state by CD measurement when dissolved in the denaturant solution, e.g., 6M guanidinium chloride(Gdn-HC1). CD also showed 100% refolding of the unfolded Lyzm by mere dilution to concentrations. Recovered enzyme activity confirmed the same result.The 6M urea + 6M LiCl solution was as effective as 6M Gdn-HCl for denaturation of Lyzm. This implies that one bifunctional denaturant was divided into two monofunctional de … More naturants. Thus urea and LiCl can be used independently to contribute the diversity of denaturant selection encountered in the protein refolding process.In the course of refolding of the fully reduced Lyzm, four basic procedures and/or conditions were established as quite useful for the refolding : 1) low concentration of protein, 2) sustain the solution at loose folding state of protein, e.g. in 2M urea, 3) delayed oxidation, or S-S bond formation after the loose folding, is recommended, 4)lower temperature throughout the process to exaggerate small energy difference between the correct structure and incorrect structure. As a result we achieved more than 95% refolding yield based on recovered activity at [Lyzm] = 1.1uM in the maturing solution.Regretfully, we have no idea by now about possible substances that enhance refolding of Lyzm. We are trying to meet the effective substances that induce refolding of the other enzymes than Lyzm. We believe that several basic knowledges established in this research work will contribute to the development of the fascinating research field of protein refolding. Less