Preparation, characterization and application of interleukin-1 beta mutant proteins with surface-accessible cysteine residues.
Two mutants of interleukin-1 beta (K27C and K138C) were produced using site-specific mutagenesis in which lysine residues at positions 27 and 138 of the mature protein sequence were substituted by cysteine residues. The conformations of the mutant proteins were studied by 1H-NMR spectroscopy and shown to be similar to the wild-type protein. The receptor-binding affinity and biological activity of K27C and K138C were also similar to wild-type protein. The substituted cysteines in both mutant proteins were shown to be solvent-accessible as judged by their reactivity towards sulfhydryl reagents. As the wild-type protein contains two cysteines, which are both solvent-inaccessible in the native state, the mutants offer the opportunity to introduce probes in a sequence-specific manner via reaction with sulfhydryl groups. Examples of this are described in which the K138C was disulfide-linked to phycobiliproteins. The highly fluorescent conjugates had similar receptor-binding affinities to that of the wild-type unconjugated protein and were found suitable for flow-cytometric analysis.
Wingfield
P
P
Glaxo Institute for Molecular Biology S.A., Geneva, Switzerland.
Graber
P
P
Shaw
A R
AR
Gronenborn
A M
AM
Clore
G M
GM
MacDonald
H R
HR
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D001667:Binding, Competitive; D003545:Cysteine; D004926:Escherichia coli; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007375:Interleukin-1 / Q000032:analysis / Q000096:biosynthesis*; D007525:Isoelectric Focusing; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010449:Peptide Mapping; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D013499:Surface Properties; D014357:Trypsin
D000596:Amino Acids; D007375:Interleukin-1; D011994:Recombinant Proteins; D014357:Trypsin; D008239:Lysine; D003545:Cysteine