High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements

Shouhei Mine; Tadashi Ueda; Yoshio Hashimoto; Yoshitsugu Tanaka; Taiji Imoto

doi:10.1016/S0014-5793(99)00332-4

High-level expression of uniformly ¹⁵N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements

Shouhei Mine, Tadashi Ueda, Yoshio Hashimoto, Yoshitsugu Tanaka, Taiji Imoto

Pharmaceutical Health Care and Sciences

Research output: Contribution to journal › Article

44 Citations (Scopus)

Abstract

The non-enzymatic deamidation of Asn to Asp is known to occur in proteins and peptides and is accelerated by phosphate buffer [Tyler-Cross, R. and Schirch, V. (1991) J. Biol. Chem. 25, 22549-22556]. We attempted to identify the site in lysozyme where a phosphate ion binds by means of ¹H-¹⁵N HSQC measurements of ¹⁵N-labeled lysozyme, which was successfully obtained using Pichia pastoris. As a result, we found that the phosphate ion was preferentially bound to Asn-103 in hen lysozyme. The method presented here may be useful for identifying the binding site of a protein with low molecular weight substances. Copyright (C) 1999 Federation of European Biochemical Societies.

Original language	English
Pages (from-to)	33-37
Number of pages	5
Journal	FEBS Letters
Volume	448
Issue number	1
DOIs	https://doi.org/10.1016/S0014-5793(99)00332-4
Publication status	Published - Apr 2 1999

All Science Journal Classification (ASJC) codes

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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10.1016/S0014-5793(99)00332-4

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Mine, S., Ueda, T., Hashimoto, Y., Tanaka, Y., & Imoto, T. (1999). High-level expression of uniformly ¹⁵N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements. FEBS Letters, 448(1), 33-37. https://doi.org/10.1016/S0014-5793(99)00332-4

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abstract = "The non-enzymatic deamidation of Asn to Asp is known to occur in proteins and peptides and is accelerated by phosphate buffer [Tyler-Cross, R. and Schirch, V. (1991) J. Biol. Chem. 25, 22549-22556]. We attempted to identify the site in lysozyme where a phosphate ion binds by means of 1H-15N HSQC measurements of 15N-labeled lysozyme, which was successfully obtained using Pichia pastoris. As a result, we found that the phosphate ion was preferentially bound to Asn-103 in hen lysozyme. The method presented here may be useful for identifying the binding site of a protein with low molecular weight substances. Copyright (C) 1999 Federation of European Biochemical Societies.",

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AU - Tanaka, Yoshitsugu

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