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.
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.
Mine
S
S
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Ueda
T
T
Hashimoto
Y
Y
Tanaka
Y
Y
Imoto
T
T
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D015870:Gene Expression; D007477:Ions; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010710:Phosphates / Q000378:metabolism*; D010843:Pichia
D007477:Ions; D009587:Nitrogen Isotopes; D010710:Phosphates; D009113:Muramidase