Stabilization of lysozyme by introducing N-glycosylation signal sequence.
We designed mutant lysozymes with N-glycosylation signal sequences (Asn48-Gly49-Thr-50 and Asn87-Ile88-Thr89) by substituting Asp to Asn at positions 48 and 87. When these mutant lysozymes were expressed by using yeast (Saccharomyces cerevisiae) in Burkholder minimum medium, N-glycosylation occurred in both lysozymes. The mutant lysozyme with the oligosaccharide at Asn87 showed a similar character to a reported polymannosyl lysozyme [Nakamura, Takasaki, Kobayashi, and Kato (1993) J. Biol. Chem. 268, 12706-12712; Kato, Takasaki, and Ban (1994) FEBS Lett. 355, 76-80]. As judged from the thermodynamic stabilities of the lysozymes obtained by the guanidine hydrochloride denaturation method, the oligosaccharide-bearing mutant lysozymes were more stable by 0.4-1.6 kcal/mol than the corresponding unglycosylated lysozymes. Therefore, it is suggested that the introduction of an N-glycosylation signal sequence into a protein is an effective means to increase the stability of the protein.
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka.
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Ueda
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Iwashita
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Hashimoto
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Imoto
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001483:Base Sequence; D004795:Enzyme Stability; D006031:Glycosylation; D019791:Guanidine; D006146:Guanidines; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship
D006146:Guanidines; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D009113:Muramidase; D019791:Guanidine