Apoplastic accumulation of the recombinant supersweet thaumatin II protein in transgenic tobacco plants
Pushin1,2 A.S., Schestibratov2 K.A., Ovchinnikova2 E.V., Shulga O.A.1, Firsov2 A.P., Dolgov1,2 S.V.
1All-Russian Institute of Agricultural Biotechnology RAAS, Moscow, Russia
2Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Russia
Introduction
Thaumatin is an intensely sweet protein isolated from the West African plant Thaumatococcus daniellii Benth (1). The molecular mass of the protein is 22 kDa and the higher order structure of the protein is maintained by eight disulfide bridges. Thaumatin is the sweetest substance for man and is about 100000 times sweeter than sucrose on a molar basis and several thousand times sweeter on a weight basis (1). Today, thaumatin is used commercially due to intense sweetness, taste masking, flavor enhancement, and synergistic properties to produce dramatic effects in food products. However, despite its advantages, industrial use of thaumatin of plant origin is very limited because of the extreme difficulty involved in obtaining the fruit from which it is extracted. Several attempts have been made to produce the recombinant protein in different expression systems (2). Recombinant thaumatin II protein in E. coli, S. lividans, S. cerevisiae and K. lactis has been expressed at low levels, with sweet properties of recombinant proteins have not been detected. More successful studies have been carried out using filamentous fungi as an alternative hosts for the expression of recombinant thaumatin. Although notable improvements in microbial expression systems for recombinant thaumatin production have been made, the industrial production of this protein are not economically feasible due to the current price of thaumatin obtained by extraction from its natural source (2). More promising approach for recombinant protein producing is a plant expression system. In this study, the lrth gene encoding the recombinant precursor of the thaumatin II protein has been constructed to evaluate the possibility of the recombinant thaumatin II production in tobacco plants.
Materials and methods
Nicotiana tabacum L. cv. Petite Havana SR1 plants were used in our work. The lrth chimeric gene and the binary vector pGD121lrTh containing this gene were performed, using the methods of molecular cloning described by Sambrook and Russell (3). The binary vector pBIThau35 with the native thauII gene was previously constructed (5). Binary vectors pGD121lrTh and pBIThau35 were transformed into Agrobacterium tumefaciens strain CBE21 as described by An et al. (4). Bacterial strains containing respective transformation vector were used to transformation of N. tabacum leaf discs according to Horsch et al. (6). Total RNA was isolated from tobacco leaves, using the method described by Gehrig et al. (7) with several modifications. The total and apoplast proteins were isolated from tobacco leaves as described by Ziegler (8) with modifications. The total and apoplast proteins from each studied line were loaded onto an SDS-PAAG gel (15% polyacrylamide) by Laemmly (9). After electrophoresis, the proteins were transferred to a NC membrane (Amersham) by tank transfer. Rabbit anti-thaumatin II polyclonal antibody was used as a primary (dilution 1:4000). Anti-rabbit IgG (dilution 1:5000) antibody conjugated with fluorescent label CY3 was used as secondary. The images of blots were obtained by Variable Mode Imager Typhoon 9200 (Molecular Dynamics, USA). Commercially available Thaumatin II (Sigma, USA) was used as a positive control. Sweetness assay was performed as described by Witty and Harvey (10) with modifications. For this assay 1 week-old tobacco leaves were used. Sweetness analysis was repeated three times.
Results and discussion
In order to achieve high level apoplastic accumulation of biologically active sweet-tasting thaumatin II protein, a chimeric gene encoding the recombinant precursor of thaumatin II protein containing N-terminal signal peptide from radish defensin RS-AP and lacking C-terminal propeptide was constructed (Fig. 1 A). The RS-AP protein N-terminal leader sequence was chosen as the target peptide because of the suggestion that it could serve as an efficient signal for secretion in plants (11). Transformed tobacco plants with thauII and lrth genes were regenerated after leaf disc transformation of N. tabacum. 12 and 16 transgenic tobacco lines harboring thauII and lrth genes respectively were obtained. All transgenic plants exhibited normal phenotypes. Three thauII and four lrth lines were randomly selected for further work. Total RNA prepared from greenhouse cultivated thauII and lrth transgenic and non-transformed plants was used as a template for RT-PCR. Single band of 454 bp corresponding to the size of the cDNA fragment encoding mature region of thaumatin II was observed in most of transgenic lines with the exception of TH10A (data not shown). Pre-mRNA transcribed from chimeric lrth gene containing 91 bp intron from radish rs-ap gene is spliced in transgenic tobacco plants. Sequence analysis of lrth cDNA demonstrated correct splicing of 91 bp intron (data not shown). Immunoblots of total protein extract from four tobacco lines transformed with lrth gene and two transgenic lines with thauII gene (Fig. 1 B) show a single band which migrates between 20 and 25 kDa. No specific band was detected in tobacco line TH10A and non-transgenic plant. Subcellular localization of LRTH and THAUII proteins was examined by comparison of the intercellular washing fluid and the remnant extract of leaves of the tobacco lines expressing thaumatin. Western-blot analysis of apoplast and remnant extracts indicate that plants transformed with the wild-type construct retain most of the produced thaumatin intracellularly. On the contrary, the most amount of thaumatin in lrth tobacco lines were detected in the intercellular washing fluid fraction (Fig. 1 C,D). Evidently the removal of the C-terminal 6 amino acids of the thaumatin II precursor and the replacement of N-terminal signal sequence caused the secretion of thaumatin into the apoplast. Biological activity evaluation of recombinant thaumatin protein using sweetness assay showed that lines TH1A, TH7A, LRTH1A, -4B, -6A, -7B had a sweeter taste distinguishable from the control and TH10A line without thaumatin II expression. Sweetness of the tobacco lines accumulating secreted thaumatin was similar to that of the lines expressing wild-type thaumatin II demonstrating conservation of the conformational state of the recombinant thaumatin.
To our knowledge, apoplastic accumulation of the biologically active thaumatin II protein in transgenic plants has been reported for the first time.
Figure 1: (A) Nucleotide and deduced amino acid sequence of a chimeric gene encoding the recombinant precursor of thaumatin II protein containing N-terminal signal peptide from radish defensin Rs-AP. The Rs-AP N-terminal signal peptide is boxed (gray), and an intron sequence is underlined. The predicted signal peptide cleavage site is indicated with an arrow. (B,C,D) Western blot analysis of transgenic tobacco lines expressing the thauII wild-type or the lrth mutant precursor construct. Lanes TH1A, -7A, -10A and LRTH1A, -4B, -6A, -7B are the thauII and lrth transgenic tobacco lines respectively, (B) 100 µg of extracted proteins per line (total extract); (C) 7 µg of protein from IWF extracts per line; (D) 100 µg of extracted proteins from remnant extracts per line (total protein).
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