Expression of the major bean proteins from Theobroma cacao (cocoa) in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae.
The production in two yeast expression systems of recombinant forms of the major proteins from the cocoa bean is described. Three major protein species are found in the cocoa bean: an albumin of molecular mass 21 kDa (p21) and two insoluble vicilin-like proteins of molecular mass 31 kDa and 47 kDa (p31 and p47, respectively). The p31 and p47 species are known to be derived from a common 67-kDa precursor (p67) by post-translational processing that includes the deletion of a hydrophilic domain located immediately after an N-terminal signal sequence. All three proteins appear to be targeted to membrane-bound storage organelles by N-terminal signal sequences. The p21 and p67 coding sequences were expressed in Hansenula polymorpha using the powerful methanol oxidase (MOX) promoter and in Saccharomyces cerevisiae using the promoter of the pyruvate kinase (PYK) gene. The expression constructs contained the native plant signal sequence, or various yeast signals. The p21 protein was successfully expressed and secreted from both yeasts. The insoluble p67 protein proved more difficult. Species of the correct molecular mass were recovered internally and small amounts of a p47 species were secreted using a yeast leader sequence. However, proteolytic cleavage, probably due to Kex2p-like processing, led to the appearance of other protein species.
Yavuz
M O
MO
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Ashton
S M
SM
Deakin
E D
ED
Spencer
M E
ME
Sudbery
P E
PE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002099:Cacao; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia; D010940:Plant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D012639:Seeds
D010940:Plant Proteins; D011994:Recombinant Proteins