Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli.
Brazzein protein comes from an edible fruit, which has a long history of being a staple in the local human diet in Africa. The attractive features of brazzein as a potential commercial sweetener include its small size (53 amino acid residues), its stability over wide ranges of temperature and pH, and the similarity of its sweetness to sucrose. Heterologous production of brazzein is complicated by the fact that the protein contains four disulfide bridges and requires a specific N-terminal sequence. Our previous protocol for producing the protein from Escherichia coli involved several steps with low overall yield: expression as a fusion protein, denaturation and renaturation, oxidation of the cysteines, and cleavage by cyanogen bromide at an engineered methionine adjacent to the desired N-terminus. The new protocol described here, which is much faster and leads to a higher yield of native protein, involves the production of brazzein in E. coli as a fusion with SUMO. The isolated protein product contains the brazzein domain folded with correct disulfide bonds formed and is then cleaved with a specific SUMO protease to liberate native brazzein. This protocol represents an important advancement that will enable more efficient research into the interaction between brazzein and the receptor as well as investigations to test the potential of brazzein as a commercially viable natural low calorie sweetener.
Assadi-Porter
Fariba M
FM
Biochemistry Department, University of Wisconsin-Madison, 171a Biochemistry Addition, 433 Babcock Drive, Madison, WI 53706, USA. fariba@nmrfam.wisc.edu
Patry
Sammy
S
Markley
John L
JL
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D010940:Plant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D025842:SUMO-1 Protein / Q000235:genetics; D013549:Sweetening Agents / Q000378:metabolism*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D013549:Sweetening Agents; C090521:brazzein protein, Pentadiplandra brazzeana