Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli

Abstract

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.

Figure 1

SDS-PAGE showing the expression of the His₆-SUMO-brazzein fusion in various host strains. Lane 1: Molecular weight markers (Invitrogen). Lanes 2/3: −IPTG / +IPTG (BL21 (DE3)/RI Codon Plus). Lanes 4/5: −IPTG / +IPTG (BL21 (DE3)/RIL Codon Plus). Lanes 6/7: −IPTG / +IPTG (BL21 (DE3)/RILP Codon Plus). Lanes 8/9: −IPTG / +IPTG (BL21(DE3)/pLysS). Lanes 10/11: −IPTG / +IPTG (Rosetta (DE3)). The His₆-SUMO-brazzein fusion is observed at 23.5 kD and brazzein at 6.4 KD.

Figure 2

Medium-scale expression and purification of His₆-SUMO-brazzein produced from BL21 (DE3)/ PRIL Codon Plus cells. Lane 1: Molecular weight markers (Invitrogen). Lane 2: −IPTG. Lane 3: total cell lysates. Lane 4: supernatant. Lane 5: pellet. Lanes 6–9: Ni-NTA column fractions (2 to 5). Lane 10: purified fusion protein. Lane 11: SUMO 1 protease cleavage product after 30 min. Lane 12: cleavage product after 60 min.

Figure 3

RP-HPLC traces of mixture resulting from SUMO 1 cleavage of the His₆-SUMO-brazzein fusion protein. (a) His₆-brazzein-SUMO fusion + 5 mM DTT. (b) His₆-brazzein-SUMO fusion + 0.1 mM DTT. Brazzein elution times are marked in folded/or unfolded/non-fully oxidized protein.

Figure 4

Spectra of the purified brazzein product obtained on a Varian 800 MHz NMR spectrometer at pH 5.2 and 25 °C. (Top) One-dimensional ¹H NMR spectrum. (Bottom) Two-dimensional ¹H-¹H NOESY map.