Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin.
Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.
Assadi-Porter
F M
FM
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Aceti
D J
DJ
Cheng
H
H
Markley
J L
JL
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000737:chemistry; D017343:Genes, Plant / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D007526:Isoelectric Point; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019685:Rosales / Q000737:chemistry*; D012995:Solubility; D013549:Sweetening Agents / Q000737:chemistry / Q000302:isolation & purification*; D013649:Taste
D003062:Codon; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013549:Sweetening Agents; C090521:brazzein protein, Pentadiplandra brazzeana; D008836:Micrococcal Nuclease