Expression, purification and initial characterization of the recombinant storage protein precursor of Theobroma cacao.
The gene encoding the 67-kDa cocoa storage protein precursor has been cloned from Theobroma cacao and expressed in Escherichia coli using the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30 degrees C using 20 mM glycine-NaOH buffer, pH 10.0, containing 1 mM oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% alpha-helix and 38% beta-sheet, in close agreement with values obtained using a secondary structure prediction program.
O'Connor
J
J
Department of Protein Engineering, BBSRC Institute of Food Research, Reading Laboratory, United Kingdom.
Revell
D F
DF
Masters
K E
KE
Connerton
I F
IF
Sumner
I G
IG
D002099:Cacao / Q000737:chemistry / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013862:Thiogalactosides / Q000378:metabolism; D013997:Time Factors
D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D013862:Thiogalactosides; D005978:Glutathione