Examination of Three Alternative Devices to Refold Correctly the Denatured Enzyme Originated from the Precipitate Produced in the Process of Protein Synthesis

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Protein refolding is one of the critical processes in the downstream of the recombinant DNA protein synthesis. The objective of the present research work is to evaluate comparatively following three devices for the correct refolding of a globular protein from its random coil structure produced, for example, in 6M GdnHCl ; that is, (1) to build the hydrophobic core in the first place, (2) to let it refold hopefully from its carboxy-terminal by immobilization at the amino-terminal, (3) to let it refold hopefully from its amino-terminal by immobilization at the carboxy-terminal.Experimentally, hydrophobic core formation in the first place, (1), was realized by use of a refolding medium of high ionic strength ; immobilization of either N or C-terminal, (2) or (3), was conducted by means of covalently bonding on sepharose gel beads according to the known methods.Of course, such irreversible covalent fixation of the protein might not meet practical uses, in protein refolding, however, it was … More adopted to get more precise evaluation for the above-mentioned three devices. Adopted globular proteins were bovine pancreatic ribonuclease A(RNase), hen egg-white lysozyme(Lyzm), and bacterial subtilisin BPN'(Sbtl).It became clear in the course of examination that RNase and Lyzm were not suitable for the present test. RNase could not be immobilized at C-terminal retaining active form due to intramolecular amidation and immobilized Lyzm was not tolerable for the generalized activity assay procedure which uses cell walls as a substrate. Fortunately, by use of Sbtl, it was possible to examine exactly the above-mentioned three devices for the correct folding.As for the device (1) which intends to form by hydrophobic core at first, we succeeded to refold 6M GdnHCl-denatured Sbtl at pH 2.4 by incubating it in 1.5-2.0M K-acetate at pH 6.5. However, the protease Sbtl suffers from autoproteolytic digestion to get a resultant maximum refolding yield of up to 30%. A precise evaluation of this device was attained by deletion of an autolysis event through the immobilization as follows.Device (2) or (3) which corresponds to the refolding of Sbtl with N-terminal or C-terminal immobilized, respectively, was conducted by repeated dissolution of immobilized Sbtl in 6M GdnHCl followed by its refolding in 2M K-acetate. In both cases, almost 100% refolding yield was achieved after the 3rd denaturation/renaturation cycle based on the recovered activity of the preceding cycle.Rate of refolding in the case of device (2) appeared to be larger than that of device (3) Moreover, a refolding medium consisted of 2M K-acetate was superior to that of 2M KCl. In conclusion, almost quantitative refolding of Sbtl was achieved in all three cases, provided that a suitable refolding environment was given. Among them, the most important device might be a hydrophobic core formation in the refolding of Sbtl followed by N-terminal and C-terminal immobilization. Less