Assignment of the disulfide bonds in the sweet protein brazzein

The thermostable sweet protein brazzein consists of 54 amino acid residues and has four intramolecular disulfide bonds, the location of which is unknown. We found that brazzein resists enzymatic hydrolysis at enzyme/substrate ratios (w/w) of 1:100-1:10 at 35–40°C for 24–48 h. Brazzein was hydrolyzed using thermolysin at an enzyme/substrate ratio of 1:1 (w/w) in water, pH 5.5. for 6 h and at 50°C. The disulfide bonds were determined, by a combination of mass spectrometric analysis and amino acid sequencing of cystine-containing peptides, to be between Cys4-Cys52, Cys16-Cys37, Cys22-Cys47, and Cys26-Cys49. These disulfide bonds contribute to its thermostability. © 1996 John Wiley & Sons, Inc.     

Monkey electrophysiological and human psychophysical responses to mutants of the sweet protein brazzein: delineating brazzein sweetness

Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all sweetness. The same pattern occurred again at the beta-turn region, where Glu41Lys gave the highest sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey sweet taste in primates.     

Expression of the sweet protein brazzein in maize for production of a new commercial sweetener

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.     

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.     

Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria

Brazzein is an intensely sweet-tasting plant protein with good stability, which makes it an attractive alternative to sucrose. A brazzein gene has been designed, synthesized, and expressed in Escherichia coli at 30 °C to yield brazzein in a soluble form and in considerable quantity. Antibodies have been produced using brazzein fused to His-tag. Brazzein without the tag was sweet and resembled closely the taste of its native counterpart. The brazzein gene was also expressed in Lactococcus lactis, using a nisin-controlled expression system, to produce sweet-tasting lactic acid bacteria. The low level of expression was detected with anti-brazzein antibodies. Secretion of brazzein into the medium has not led to significant yield increase. Surprisingly, optimizing the codon usage for Lactococcus lactis led to a decrease in the yield of brazzein.     

Chemical synthesis and characterization of the sweet protein mabinlin II

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste. © 1998 John Wiley & Sons, Inc. Biopoly 46: 215–223, 1998     

Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts

Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin).     

Structure‐function relationships of brazzein variants with altered interactions with the human sweet taste receptor

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non‐carbohydrate sweeteners. In an investigation of sequence‐dependent functional properties of the protein, we used NMR spectroscopy to determine the three‐dimensional structures and dynamic properties of two Brz variants: one with a single‐site substitution (D40K), which is three‐fold sweeter than wild‐type Brz, and one with a two‐residue insertion between residues 18 and 19 (ins₁₈RI₁₉), which is devoid of sweetness. Although the three‐dimensional folds of the two variants were very similar to wild‐type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter‐stand H‐bond between the side chains of residues Gln46 and Asp40 present in wild‐type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G‐protein coupled human sweet receptor. On the other hand, the Arg‐Ile insertion within Loop9–19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.     

Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in Kluyveromyces lactis

Abstract

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25?°C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80?°C for 1?h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50?mM NaCl in 50?mM sodium acetate buffer (pH 4.0) as a wash buffer and 400?mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.     

Synthesis and characterization of gold glyconanoparticles functionalized with sugars of sweet Sorghum syrup

Gold glyconanoparticles were synthesized by a simple, rapid, and eco-friendly method by using sweet Sorghum syrup for application in biomedicine and biotechnology. The nanostructures of the prepared gold nanoparticles were confirmed by using UV-visible absorbance, TEM, SAED, FTIR, EDAX, XRD, and photoluminescence analyses. The formation of gold nanoparticles at both room and boiling temperatures and kinetics of the reaction were monitored by UV-visible spectroscopy and TEM studies. TEM analysis revealed that the obtained nanoparticles were mono-dispersed and spherical in shape with an average particle size of 7 nm. The size of the nanoparticles was influenced by the concentration of Sorghum syrup. The presence of elemental gold was confirmed by EDAX analysis. Based on the FTIR analysis, it was observed that the sugars present in the Sorghum syrup possibly acts as capping agents. The zeta potential analysis revealed that the glyconanoparticles were negatively charged with a potential of -25 mV. The XRD and SAED patterns also suggest that the nanoparticles were crystalline in nature and these particles were found to exhibit visible photoluminescence. Fructose and glucose present in sweet Sorghum syrup were demonstrated as responsible sugars for the reduction of gold ions, and sucrose stabilized the formed nanoparticles. The proposed mechanism for the formation and stabilization of gold glyconanoparticles is based on the phenomenon of "macromolecular crowding." This is the first report on the use of sweet Sorghum syrup for the green synthesis of gold glyconanoparticles at both room and boiling temperatures.     

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

Critical regions for the sweetness of brazzein

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29-33 and 39-43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.     

植物甜蛋白brazzein基因的克隆与表达

根据大肠杆菌偏爱的密码子,利用PCR技术体外人工合成brazzein cDNA序列,并将其克隆至原核高效表达载体pET30a( )中。重组载体pET30a( )-brazzein转化至大肠杆菌BL21(DE3)中,经IPTG诱导后,SDS-PAGE结果证明pET30a( )-brazzein在大肠杆菌中获得高效表达,目的蛋白占总菌体蛋白25％左右。     

Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein

Brazzein, originally isolated from the fruit of the African plant Pentadiplandra brazzeana Baillon, is the smallest, most heat-stable and pH-stable member of the set of proteins known to have intrinsic sweetness. These properties make brazzein an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. We have used the three-dimensional structure of the protein (J. E. Caldwell et al. (1998) Nat. Struct. Biol. 5, 427-431) as a guide in designing 15 synthetic genes in expression constructs aimed at delineating the sweetness determinants of brazzein. Protein was produced heterologously in Escherichia coli, isolated, and purified as described in the companion paper (Assadi-Porter, F. M., Aceti, D., Cheng, H., and Markley, J. L., this issue). Analysis by one-dimensional (1)H NMR spectroscopy indicated that all but one of these variants had folded properly under the conditions used. A taste panel compared the gustatory properties of solutions of these proteins to those of sucrose and brazzein isolated from fruit. Of the 14 mutations in the des-pGlu1-brazzein background, four exhibited almost no sweetness, six had significantly reduced sweetness, two had taste properties equivalent to des-pGlu1-brazzein (two times as sweet as the major form of brazzein isolated from fruit which contains pGlu1), and two were about twice as sweet as des-pGlu1-brazzein. Overall, the results suggest that two regions of the protein are critical for the sweetness of brazzein: a region that includes the N- and C-termini of the protein, which are located close to one another, and a region that includes the flexible loop around Arg43.     

Interaction of sweet proteins with their receptor. A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin.

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of "sweet fingers", i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential "sweet fingers" of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37-47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the "wedge model", recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett.526, 1-3.].     

Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.     

Synthesis and structural characterization of human-identical lung surfactant SP-C protein

An efficient synthesis for human-identical lung surfactant protein SP-C is described with a semi-automated solid phase synthesizer using Fmoc chemistry. Double coupling and acetic anhydride capping procedures were employed for synthetic cycles within the highly hydrophobic C-terminal domain of SP-C. Isolation of the protein was performed by mild cleavage and deprotection conditions and subsequent HPLC purification yielding a highly homogeneous protein as established by sequence determination, electrospray, plasma desorption and MALDI mass spectrometry. A general method has been employed for the preparation of Cys-palmitoylated protein by using temporary Cys(tButhio) protection, in situ deprotection with β-mercaptoethanol and selective palmitoylation of resin-bound SP-C. The mild synthesis and isolation conditions provide SP-C with a high α-helical content, comparable to that of the natural SP-C, as assessed by CD spectra. Furthermore, first biophysical data indicate a surfactant activity comparable to that of the natural protein. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Correlation of the sweetness of variants of the protein brazzein with patterns of hydrogen bonds detected by NMR spectroscopy

In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (trans-hydrogen bond couplings). We have applied this approach to screen a series of five single site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long range, three-dimensional data correlating connectivities among backbone 1HN, 15N, and 13C' atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three "sweet" variants exhibited the same pattern of hydrogen bonds, whereas all three "non-sweet" variants lacked one hydrogen bond at the middle of the alpha-helix, where it is kinked, and one hydrogen bond in the middle of beta-strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.