Characterization of asparagine deamidation and aspartate isomerization in recombinant human interleukin-11.
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.
Zhang
Wei
W
Wyeth BioPharma, Genetics Institute Campus, Andover, Massachusetts 01810, USA. wxzhang@wyeth.com
Czupryn
J Marta J
JM
Boyle
Philip T
PT
Amari
John
J
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017370:Interleukin-11 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods
D000577:Amides; D017370:Interleukin-11; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine