Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding.
Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (Il2) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined. All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold-improved renaturation yield. One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and high-affinity receptor binding. The binding to the low-affinity Il2-binding protein (Tac antigen) was unimpaired. A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high- and the low-affinity receptor, however, was reduced about 20-fold. Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins. It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.
Weigel
U
U
Physiologisch-Chemisches Institut der Universität Würzburg.
Meyer
M
M
Sebald
W
W
D000818:Animals; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D006146:Guanidines; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D004247:DNA; D019791:Guanidine