重组人白细胞介素-11中天冬酰胺脱酰胺作用和天冬氨酸异构化的表征
Characterization of asparagine deamidation and aspartate isomerization in recombinant human interleukin-11.
作者信息
Zhang Wei, Czupryn J Marta J, Boyle Philip T, Amari John
机构信息
Wyeth BioPharma, Genetics Institute Campus, Andover, Massachusetts 01810, USA.
出版信息
Pharm Res. 2002 Aug;19(8):1223-31. doi: 10.1023/a:1019814713428.
UNLABELLED
PURPOSE; The aim of this study was to investigate asparagine (Asn) deamidation and aspartate (Asp) isomerization and to measure the content of isoaspartate (isoAsp) in recombinant human interleukin-11 (rhIL-11).
METHODS
The rhIL-11 control and heat stressed samples were characterized with trypsin and endoproteinase Asp-N peptide mapping, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI MS) and capillary electrophoresis (CE). The total isoAsp content and bioactivity were also assessed.
RESULTS
Stress of rhIL11 at 30 degrees C for 6 weeks in liquid resulted in significant isomerization of Asp45 and Asp47. Isomerization of Asp51 and deamidation of Asn49 were also detected at low levels. The stressed rhIL-11 molecule contained 0.3 mol of isoAsp per mol of protein, compared to only 0.007 mol/mol of protein in the control.
CONCLUSIONS
Asp and Asn residues, located in a loop structure of rhIL-11, undergo isoAsp formation under stressed conditions.
未标记
目的；本研究旨在研究重组人白细胞介素-11（rhIL-11）中天冬酰胺（Asn）的脱酰胺作用和天冬氨酸（Asp）的异构化，并测定异天冬氨酸（isoAsp）的含量。
方法
采用胰蛋白酶和天冬氨酸-N内肽酶肽图分析、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、反相高效液相色谱（RP-HPLC）、电喷雾电离质谱（ESI MS）和毛细管电泳（CE）对rhIL-11对照样品和热应激样品进行表征。还评估了总异天冬氨酸含量和生物活性。
结果
rhIL11在30℃液体中应激6周导致Asp45和Asp47发生显著异构化。还检测到低水平的Asp51异构化和Asn49脱酰胺作用。应激的rhIL-11分子每摩尔蛋白质含有0.3摩尔异天冬氨酸，而对照中每摩尔蛋白质仅含0.007摩尔/摩尔。
结论
位于rhIL-11环结构中的Asp和Asn残基在应激条件下会形成异天冬氨酸。
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