Construction of a General Principle for Increasing Protein ThermostAbility ; Verification of The Proline Theory

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This research project has been programmed to establish a general principle of increasing protein thermostability, conforming a "proline theory".The genes of oligo-1, 6-glucosidase were cloned individually from three Bacillus strains (B. cereus ATCC7064 (I), B. coagulans ATCC7050 (II), B. thermoglucosidasius KP1006 (III)) growing at a different range of temperature. The primary structures of those homologous enzymes deduced from the nucleotidesequences were determined to be compared. Those revealed that proline residues of each enzyme increased with thermostability ((I)19-(II)23-(III)32) and most of prolines occurred only in thermostable enzymes ((II) and (III)) were included in the loop structures. Three residues (Lys121, Glu175, Glu290) of B. cereus enzyme, substituted by proline in two other enzymes, were replaced with proline by site-directed mutagenesis. The wild and mutant enzymes constructed (Mnt-1=Lys121- P ; Mut-2=Lys121- P, Glu175- P ; Mut-3=Lys121- P, Glu175- P, Glu290- P) sh … More owed that temperatures for 50% inactivation by 10 min holding were 44.5, 45.9, 46.3, 47.1, ^ﾟC, respectively. When each enzyme was treated at 45 ^ﾟC, time for 50% inactivation was 6 min for the wild, 27 min for Mut-1, 120 min for Mut-2, and much longer than 120 min for Mut-3. These results proved that replacements with proline on the loop should be effective to enhance cumulatively thermostability of a protein and the above principle should become practical.It becomes necessary to determine the exact positions of proline residues in the tertiary structure of oligo-1, 6-glucosidase. For the purpose, B. cereus enzyme was overproduced under the control of lac promoter in Escherichia coli and extensively purified for crystallization and subsequen X-ray analysis. The enzyme was crystallized by the hanging drop method using ammonium sulfate as precipitant. The preliminary data of X-ray analysis were obtained and further analysis is going on in collaboration with a group of Institute for Protein Research, Osaka University. Less