Permuteins of interleukin 1{beta}--a simplified approach for the construction of permutated proteins having new termini |
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Authors: | Horlick RA; George HJ; Cooke GM; Tritch RJ; Newton RC; Dwivedi A; Lischwe M; Salemme FR; Weber PC; Horuk R |
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Affiliation: | 1The Du Pont Merck Pharmaceutical Co. PO Box 80400, Du Pont Experimental Station, Wilmington, DE 19880-0400
3 Glenolden, PA 19036
4Sterling Winthrop Pharmaceutical Research Division 9 Great Valley Pkwy, Malvern, PA 1935
5Department of Protein Chemistry Genentech, 460 Point San Bruno Blvd, South San Francisco, CA 94080, USA |
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Abstract: | A technique for the rapid and simple generation of permutatedversions of the interleukin-1ß (IL-1ß) geneis described. In this method, the human IL-1ß cDNAis twice amplified by the polymerase chain reaction (PCR) andthe resulting DNA fragments are ligated in tandem. Between thetwo genes, the DNA sequence encodes a short four amino acidloop to link the native N- and C-terminal ends of the IL-1ßprotein. By using PCR amplification from this starting template,a new version of the IL-1ß cDNA was obtained thatencodes a permutated form of the IL-1ß protein wherethe new N- and C-terminal amino acids correspond to residues65 and 64 of the native IL-1ß sequence, respectively.The name permutein is proposed to describe proteinsgenerated by this technology. The molecular profile (IL-1 receptorbinding, biologic activity and solution properties) of the IL-1permutein produced by this technology, permutein 65/64, is shownto be identical to that of native IL-1ß The approachshould be useful to define further the structural features ofthis protein that are important for its function. |
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Keywords: | gene assembly/ interleukin/ in vitro mutagenesis/ PCR/ permutein |
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