Table 2.
Extraction, isolation, purification and characterization methods of the wild-type, synthetic and recombinant monellin. Wild-type monellin is referred to as the monellin extracted from Dioscoreophyllum cumminsii Diels. * MNEI monellin is a single-chain monellin composed of the two chains of naturally occurring monellin linked by a Gly-Phe dipeptide.
| Source | Extraction | Isolation | Purification | Characterization | Main Results | Ref. |
|---|---|---|---|---|---|---|
| D. cumminsii | - | Salt precipitation | Ion-exchange chromatography | SDS- PAGE; Gel filtration; Fluorescence spectroscopy. |
The molecular weight of monellin obtained by SDS-PAGE and gel filtration is 10.5 KDa and 10.0 KDa, respectively. The primary structure has 91 amino acids with single residues of tryptophan, methionine, and cysteine |
[54] |
| D. cumminsii | - | Salt precipitation; Gel filtration on Sephadex G-50 (mobile phase: 1% aqueous acetic acid). |
Ion-exchange chromatography; Affinity chromatography (mobile phase: 6 M guanidine. HCl, 0.1 M sodium phosphate buffer, pH 7.4, containing 0.2 M dithiothreitol). |
SDS-PAGE; Edman degradation. |
Monellin is composed of two chains of similar length linked by non-covalent bonds. However, the subunits, devoid of sweetness, are not identical. By abolishing the thiol group, monellin loses its sweet taste |
[58] |
| Standard | - | - | Ion-exchange chromatography using a Sephadex-CM 25-gel (mobile phase: 100 mM NaCl, 10 mM phosphate buffer); RT-HPLC (gradient: 30% methanol, 0.1% TFA to 70% methanol, 0.1% TFA). |
N-terminal amino acid sequencing; ESI-MS/MS; Size-exclusion chromatography; NMR; CD |
The secondary structure: in the native state, the chain A of monellin consists of β-structure, and chain B contains both α and β-structures; Addition of 50% ethanol or TFE denatures the protein. |
[60] |
| Synthesized | - | - | RT-HPLC; HIC. |
HPLC; ESI-MS; Quantitative amino acid analysis. |
Monellin contains five Aspartate residues and nine Lysine residues; Asp87 h plays an important role in monellin sweetness. |
[61] |
| MNEI monellin * expressed in E. coli | - | - | Ion-exchange chromatography using a HiPrep26/60 Sephacryl 100 column (mobile phase: sodium acetate and sodium chloride); Gel filtration in a G-75 column (elution: 150 mM ammonium bicarbonate). |
X-ray Crystallography (resolution of 1.15 Ă) | The crystal contains a single MNEI protein in the asymmetric unit and lacks the dimer interface observed in all the previous crystal structures of monellin and its single-chain derivatives; Four stably bound negative ions are also located and can be related to potential electrostatic interactions with the surface of the sweet taste receptor; |
[56] |
| MNEI and muted MNEI monellin expression in E. coli | - | - | Ion-exchange chromatography; Size-exclusion chromatography. |
SDS-PAGE; CD. |
Mutated protein eluted at high salt concentrations (200 mM NaCl) relative to MNEI monellin (100–150 mM NaCl). CD spectra of the mutated monellin presents two minimums at 201 and 213 nm; At pH 2.5–6.8, the β-sheet and α-helix content exhibit minor changes, which corroborates the folding stability of the mutated monellin |
[59] |
| E. coli | - | - | Ion exchange chromatography; Size-exclusion chromatography. |
ESI-MS; Hydrogen exchange-mass spectrometry. |
Monellin purity exceeds 95%. Chain A and B molar masses are 5.382 KDa and 5.965 KDa, respectively; Double-chain monellin (dcMN) unfolds in a barrier-limited manner in which chain B undergoes non cooperative exchange and chain A cooperatively. |
[62] |
| E. coli | - | Incubation of the cell extract at 60 °C for 10 min and at pH 4 for 1 h at 4 °C | Ion-exchange chromatography using a Sephadex CM-50 column (gradient: 0 to 0.4 M NaCl); SDS-PAGE. |
- | Recombinant monellin yield of 43 mg/g of dry cell wt. Purity confirmed by SDS-PAGE. |
[63] |
| Candida utilis | - | - | Ion exchange chromatography using a CM-Sepharose column (gradient: 0–0.4 M NaCl); | SDS-PAGE | Molecular mass of single-chain protein is 10.0 KDa. In total soluble protein, 5% is monellin. |
[64] |
| E. coli | - | - | Ion-exchange chromatography using CM-cellulose and DEAE-cellulose columns due to the nature of the different mutants proteins; RT-HPLC using a Resource RPC column (mobile phase: eluent A composed of 10% acetonitrile/water with 0.1% TFA, and eluent B of 90% acetonitrile/water with 0.1% TFA; gradient: 20–50% B). |
SDS-PAGE; Amino acid analysis followed by hydrolysis; MALDI-TOF-MS; NMR-HSQC spectroscopy; Fluorescence emission spectroscopy; CD. |
The intermolecular and intramolecular Coulombic interactions are involved in the stabilization of recombinant monellin and in the reconstitution of the wild-type monellin. Charge interactions may significantly modulate the folding of monellin and its binding to the sweet receptors by modifying association rates. |
[65] |
| E. coli | - | - | Nickel-affinity chromatography; RT-HPLC (mobile phase: acetonitrile 5%; flow rate of 1 mL/min). |
SDS-PAGE | H-Monellin shows an identical fold and a typical β-sheet-rich structure. The molar mass of H-monellin is 16.0 KDa, with 14.0 KDa for the MNEI monellin. |
[66] |
TFE: trifluoroethanol; HIC: hydrophobic interaction chromatography.