Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris

Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin.     

Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris

Thaumatin is a sweet-tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the nucleotide sequence of thaumatin I has been controversial. We have cloned two thaumatin cDNAs from the fruit of Thaumatococcus daniellii Benth. One is the same nucleotide sequence as that of thaumatin II already reported, and the other is a novel nucleotide sequence. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin I, the only exception being the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaumatin I cDNA was transformed into Pichia pastoris X-33, and the recombinant thaumatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaumatin I was the same as that of the plant thaumatin I, although several unexpected amino acid residues were attached to the N-terminal of the recombinant thaumatin I. These indicate that the N-terminal portion of thaumatin is not critical for the elicitation of sweetness.     

Transgenic tomato plants as supersweet protein thaumatin II producers

Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.     

Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.     

Purification of chitinolytic protein from Rehmannia glutinosa showing N-terminal amino acid sequence similarity to thaumatin-like proteins.

We have purified a 21-kDa protein, designated as P1, from Rehmannia glutinosa to homogeneity by ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, and preparative native PAGE. The purified P1 had chitin degradation activity. The N-terminal amino acid sequence of P1 indicated that it is very similar to those of thaumatin and other reported thaumatin-like proteins.     

Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans.

A bioassay using Phytophthora infestans was developed to determine whether inhibitory proteins are induced in pathogen-inoculated plants. Using this bioassay, AP24, a 24-kilodalton protein causing lysis of sporangia and growth inhibition of P. infestans, was purified from tobacco plants inoculated with tobacco mosaic virus. Analysis of the N-terminal amino acid sequence identified AP24 as the thaumatin-like protein osmotin II. The sequence was also similar to NP24, the salt-induced protein from tomato. Subsequently, we purified a protein from tomato plants inoculated with P. infestans that had inhibitory activities identical to those of the tobacco AP24. The N-terminal amino acid sequence of this protein was also similar to those of osmotin and NP24. In general, both the tobacco and tomato AP24 caused lysis of sporangia at concentrations greater than 40 nanomolar and severely inhibited hyphal growth at concentrations greater than 400 nanomolar. Because both proteins were induced by pathogen inoculation, we discussed the possible involvement of these proteins as a plant defense mechanism.     

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.     

Expression of synthetic thaumatin genes in yeast

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.     

Purification and characterization of novel transglutaminase from Bacillus subtilis spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37 degrees C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60 degrees C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked epsilon-(gamma-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as alphas-casein and BSA.     

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the -galactosidase leader peptide, under the control of the -galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Purification and characterization of a soluble form of rat liver NADPH-cytochrome P-450 reductase highly expressed in Escherichia coli

A recombinant cDNA of rat liver NADPH-cytochrome P-450 reductase (CPR), which lacks the N-terminal hydrophobic region, was amplified by PCR and cloned. The N-truncated cDNA named tCPR was ligated into a pBAce vector and expressed. The tCPR protein expressed in Escherichia coli was recovered into the soluble fraction of the cell lysate and purified to homogeneity by three sequential purification procedures; (I) anion-exchange chromatography on a DEAE-cellulose (DE-52) column, (II) affinity chromatography on 2('),5(')-ADP Sepharose 4B, and (III) chromatography on a hydroxyapatite column. The average yield was 47mg per liter of culture medium. The absorption spectrum of the purified tCPR protein was identical to that of a native full-length CPR purified from rat liver, indicating that tCPR also possesses one molecule each of FAD and FMN. The tCPR protein was able to reduce cytochrome c and was also able to assist heme degradation by a soluble form of rat heme oxygenase-1. However, it failed to support the O-deethylation of 7-ethoxycoumarin by cytochrome P-450 1A1, indicating that the presence of the N-terminal hydrophobic domain is necessary for CPR to interact with cytochrome P-450. Previously, to prepare a soluble form of CPR, full-length CPR was treated with proteinases that selectively removed the N-terminal domain. With the expression system established in this study, however, the soluble and biologically active tCPR protein can be readily prepared in large amounts. This expression system will be useful for mechanistic as well as structural studies of CPR.     

Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas

Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60°C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD.     

Crystal structure of the sweet-tasting protein thaumatin II at 1.27Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 ?. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.     

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as α_s-casein and BSA.     

Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.