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. 2023 May 10;12(10):1943. doi: 10.3390/foods12101943

Table 1.

Extraction, isolation, purification and characterization methods of the wild-type, synthetic and recombinant brazzein. Wild-type brazzein is referred to as the brazzein extracted from P. brazzeana.

Source Extraction Isolation Purification Characterization Main Results Ref.
P. brazzeana 0.1 M phosphate buffer at pH 7.0 containing 5% glycerol, 0.1 mM DTT, 20 mL PMSF, 0.1 mM EDTA and 0.5% (w/v) PVP at 4 °C Protein precipitation with ammonium sulfate 30% and 85% Ion-exchange chromatography using a CM-Sepharose CLdB column (gradient: NaCl of 0.1 to 0.4 M in 20 mM sodium citrate at pH 3.6) SDS- PAGE;
ESI-MS;
sequence determination by S-Pyridylethylation and S-carboxymethylation of brazzein and peptide fragment separation by RT-HPLC.		 Brazzein is a single-chain polypeptide; the molecular weights obtained by SDS-PAGE and ESI-MS were 6.5 KDa and 6.473 KDa, respectively;
C-terminal is a tyrosine;
8 cysteines out of 54 residues.		 [34]
Nicotiana tabacum cv. Xanthi 0.1 M phosphate buffer at pH 7.0 containing 5% glycerol, 0.1 mM DTT, 20 ml PMSF, 0.1 mM EDTA and 0.5% (w/v) PVP at 4 °C Protein precipitation with ammonium sulfate 30% and 85% C18 RT-HPLC 1H NMR The secondary structure: 1 α-helix, one short 310 -helix, two strands of antiparallel β-sheet, and probably a third strand near the N-terminal;
The core of the brazzein structure is a “cysteine-stabilized alpha-beta” (CSαβ) motif;
The tertiary structure stabilized by four disulfide bonds.		 [35]
P. brazzeana Buffer solution (40 mM Tris-HCl, 50 mM NaCl, 20 mM EDTA, 55 mM sodium citrate, and 12 mM sodium thiosulfate, pH 6.7) Ammonium sulfate 30–80% precipitation; heat treatment (80 °C for 2 h) DEAE-Sepharose anion-exchange chromatography (gradient: 0 to 1.0 M NaCl in 20 mM Tris-HCl at pH 8.0); CM-Sepharose cation-exchange chromatography (gradient: 50 mM sodium acetate buffer with 400 mM NaCl at pH 4.0) SDS-PAGE;
RP-HPLC;
CD;
N-terminal amino acid sequencing;
ESI-MS/MS.		 The expressed brazzein presents a molar mass of 6.5 KDa;
Elution time on RT-HPLC is identical to the brazzein expressed from K. lactis;
The secondary structure: 9.9% of α-helices and 19.7% of β-sheets.		 [36]
P. brazzeana 0.1 M phosphate buffer at pH 7.0 containing 5% glycerol, 0.1 mM DTT, 20 mL PMSF, 0.1 mM EDTA and 0.5% (w/v) PVP at 4 °C Protein precipitation with ammonium sulfate 30% and 85% Ion-exchange chromatography using a CM-Sepharose CLdB column (gradient: NaCl of 0.1 to 0.4 M in 20 mM sodium citrate at pH 3.6) 1H NMR (pH 5.2; 22 °C) Folding is due to the ‘cysteine-stabilized alpha-beta’ (CSαβ) motif stabilizing the α-helix by two disulfide bonds with the nearest β-strand;
Total of four disulfide bonds responsible for protein folding and its sweetness.		 [37]
P. brazzeana 0.1 M phosphate buffer at pH 7.0 containing 5% glycerol, 0.1 mM DTT, 20 mL PMSF, 0.1 mM EDTA and 0.5% (w/v) PVP at 4 °C Protein precipitation with ammonium sulfate 30% and 85% HPLC (mobile phase composed of 0.05% TFA (A) and acetonitrile with 0.05% TFA gradient: 10% B to 18% B obtained in 55 min, 18% B to 25% B in 65 min and 25% B to 10% B in 75 min; flow rate of 10 mL/min) X-ray Crystallography (pH 4; 293 K) to 1.8 Ă resolution The first brazzein crystal to 1.8 Ă resolution is reported. [38]
Escherichia coli (E. coli) 0.1 M phosphate buffer at pH 7.0 containing 5% glycerol, 0.1 mM DTT, 20 mL PMSF, 0.1 mM EDTA and 0.5% (w/v) PVP at 4 °C Protein precipitation with ammonium sulfate 30% and 85%
HPLC (mobile phase composed of 0.05% TFA (A) and acetonitrile (B) with 0.05% TFA gradient: 10% B to 18% B obtained in 55 min, 18% B to 25% B in 65 min and 25% B to 10% B in 75 min; flow rate of 10 mL/min) X-ray Crystallography (pH 4; 293 K) to 1.8 Ă resolution The crystal structure is composed of one short α -helix and three β-strands which form a triple-stranded antiparallel β–sheet; it also contains an additional α-helix that is absent in the brazzein solution structure;
In solution, brazzein exists as a monomer;
In crystal, brazzein forms a homodimer stabilized by six hydrogen bonds.		 [39]
E. coli - - Nickel-affinity chromatography (mobile phase: 0–500 mM Imidazol in PBS)
Cation-exchange chromatography (SP-Sepharose column; gradient: 30–1000 mM NaCl)		 X-ray Crystallography (pH 4–4.5; 291 K) Structures of the recombinant brazzein exhibit two α -helices and three β-strands linked by four disulfide bonds with a significantly altered electrostatic distribution on the surface. [40]
E. coli Tris–HCl buffer 50 mM (pH 8.0, with 2 mM EDTA) - CM-cellulose ion-exchange chromatography (mobile phase: 50 mM Tris-HCl with 0.6 M NaCl, pH 7.6);
RT-HPLC		 NMR (25 °C) The recombinant protein adopts a cysteine-stabilized αβ (CSαβ) fold stabilized by 17 inter-strand α-helical hydrogen bonds and four disulfide bridges, that together contribute to the marked heat (100 °C) and cold (216 °C) stability of brazzein within a pH range of 2.5–11.0. [41]
E. coli Tris-HCl buffer 50 mM (pH 8.0, with 2 mM EDTA) - CM-cellulose ion-exchange chromatography (mobile phase: 50 mM Tris-HCl with 0.6 M NaCl, pH 7.6);
RT-HPLC		 RT-HPLC;
NMR (pH 5.2; 37 °C).		 Compared to the wild-type protein, the mutated brazzein displays an extended β-structure due to the terminal β-sheets and increased dynamics. [42]
Kluyveromyces lactis - - Nickel-affinity chromatography CD (pH 7.6, 25 °C);
Intrinsic fluorescence;
ANS fluorescence.		 Recombinant proteins (E9K and E9G) presented a molar mass of 6.5 KDa;
The secondary structure of E9K brazzein is stabler than E9K and the wild-type brazzein;
Brazzein has 6 tyrosine residues at positions 8, 11, 24, 39, 51 and 54, and a phenylalanine residue at position 38;
The tertiary structure of the recombinant proteins is more compact than the wild-type brazzein;
The local tertiary structure around tyrosine residues in the wild-type brazzein and E9G brazzein is more exposed to a polar environment;		 [43]
Pichia pastoris - - CM-Sepharose cation-exchange chromatography (gradient: 100 to 1000 mM NaCl IN 50 mM sodium acetate, at pH 4.0; flow rate of 1 mL/min) SDS-PAGE;
RT-HPLC (mobile phase: 0.1% TFA and 70% acetonitrile with 0.1% TFA);
CD (25 °C).		 104 mg/L can be obtained from the recombinant brazzein;
The molar mass of recombinant protein is 6.5 KDa with an elution time in RT-HPLC of 9 ± 0.5 min;
Compared to the wild-type brazzein, no significant alterations in the secondary structure of recombinant brazzein are observed by CD analysis.		 [44]
Bacillus licheniformis - - Cation-exchange chromatography (SP-Sepharose column; gradient: 0 to 1 M NaCl in 50 mM sodium acetate buffer pH 4 in 50 min; flow rate of 1 mL/min) SDS-PAGE;
ESI-MS;
NMR.		 Recombinant proteins are correctly folded; [45]
E. coli - - Cation-exchange chromatography (Q-Sepharose column; gradient: 300 to 1000 mM NaCl in 20 mM Tris buffer; flow rate of 1 mL/min) SDS-PAGE;
LC-MS/MS;
CD (25 °C).		 85% purity; formation of disulfide bonds is confirmed by LC-MS/MS;
The secondary structure of the recombinant protein is similar to the wild-type brazzein.		 [46]
E. coli - - Nickel-affinity chromatography SDS-PAGE;
LC-MS/MS;
MALDI-TOF.		 Formation of disulfide bonds is confirmed by LC-MS/MS;
The secondary structure of the recombinant protein is similar to the wild-type brazzein		 [46]
Lactococcus lactis Talon-affinity chromatography;
RT-HPLC.		 Edman degradation;
SDS-PAGE.		 The primary structure of the recombinant brazzein is similar to the wild-type brazzein. [47]
Talon-affinity chromatography;
RT-HPLC.		 Edman degradation;
SDS-PAGE.		 The primary structure of the recombinant brazzein is similar to the wild-type brazzein. [47]

DTT: dithiothreitol; PMSF: phenyhnethylsulfonyl fluoride; EDTA: ethylenediamine tetra acetic acid; PVP: polyvinylpolypyrrolidone; CM: carboxymethyl; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC: reverse-phase high-performance liquid chromatography; ESI-MS: electrospray ionization mass spectrometry; NMR: Nuclear Magnetic Resonance; TFA: trifluoroacetic acid; SP: sulfopropyl; ANS: 8-Anilino naphthalene 1-sulfunate; DEAE: diethylaminoethyl.