Biotinylation of reactive amino groups in native recombinant human interleukin-1 beta.
Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1 beta products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92-94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1 beta by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1 beta was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1 beta.
Yem
A W
AW
Department of Biopolymer Chemistry, Upjohn Company, Kalamazoo, Michigan 49001.
Zurcher-Neely
H A
HA
Richard
K A
KA
Staite
N D
ND
Heinrikson
R L
RL
Deibel
M R
MR
D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D007375:Interleukin-1 / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013388:Succinimides
D007375:Interleukin-1; D011994:Recombinant Proteins; D013388:Succinimides; C053827:biotinyl-N-hydroxysulfosuccinimide ester; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D001710:Biotin