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 encode a series of peptides comprising perfect repeat motifs based on a high molecular 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 Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.
Feeney
K A
KA
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, BS41 9AF, Bristol, UK.
Tatham
A S
AS
Gilbert
S M
SM
Fido
R J
RJ
Halford
N G
NG
Shewry
P R
PR
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004396:Coloring Agents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D005983:Glutens / Q000031:analogs & derivatives* / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics / Q000502:physiology*; D012639:Seeds; D014908:Triticum
D004396:Coloring Agents; D010446:Peptide Fragments; D012333:RNA, Messenger; D005983:Glutens; C037302:glutenin