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Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification
Authors:JC Smith  RB Derbyshire  E Cook  L Dunthorne  J Viney  SJ Brewer  HM Sassenfeld  LD Bell
Institution:Searle Research and Development, P. 0. Box 53, Lane End Road, High Wycombe, Buckinghamshire U.K.. Tel. (0494) 21124
Abstract:A 43-bp DNA duplex coding for poly(arginine) poly(arg)] has been synthesised by modified phosphotriester procedures. It has been inserted into the BglII and BamHI restriction sites of a cloned synthetic β-urogastrone (Uro) gene, under the control of the trp promoter. Subsequent induction with 3β-indole acrylic acid produces β-Uro with a C-terminal poly(arg) fusion. The raised isoelectric point of this polypeptide fusion facilitates rapid purification by cation exchange chromatography. The C-terminal poly(arg) tail can be readily removed by treatment with carboxypeptidase B.
Keywords:Recombinant DNA  urogastrone  expression plasmid  protein stability  carboxypeptidase  Ap  ampicillin  bp  base pairs  CNBr  cyanogen bromide  DTT  dithiothreitol  HPLC  high-performance liquid chromatography  IAA  3β-indole acrylic acid  PAGE  polyacrylamide gel electrophoresis  poly(arg)  polyarginine  SDS  sodium dodecyi sulphate  Uro  urogastrone
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