Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that enco de a series of peptides comprising perfect repeat motifs based on a high mo lecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Es cherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to sh ow any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclo nal antibody showed the presence of the R6 peptide (M-r 22 005) in the crud e cell extracts and both this and the R3 peptide (M-r, 12 005) were subsequ ently purified by extraction with hot aqueous ethanol followed by precipita tion with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blu e R250 or other protein stains only weakly and this was considered to accou nt for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptide s had similar p-turn rich structures similar to the repetitive sequences pr esent in the whole HMW glutenin subunits. We conclude that expression of pe rfect repeat peptides in E. coli is a suitable system for the study of stru cture-function relationships in wheat gluten proteins and other highly repe titive proteins. (C) 2001 Elsevier Science B.V. All rights reserved.