Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini.
A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.
Horlick
R A
RA
Du Pont Merck Pharmaceutical Co., Wilmington, DE 19880-0400.
George
H J
HJ
Cooke
G M
GM
Tritch
R J
RJ
Newton
R C
RC
Dwivedi
A
A
Lischwe
M
M
Salemme
F R
FR
Weber
P C
PC
Horuk
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007375:Interleukin-1 / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000138:chemical synthesis
D007375:Interleukin-1; D011994:Recombinant Proteins