Fraley (1988). Total DNA (10 I-tg) was digested with Band-tl or EcoRI, electrophoresed through. 0.7% agarose gels, and transferred to MSI membranes (Micron ...
BIOTECHNOLOGY LETTERS Volume 17 No.12 (Dec.1995) p.1279-1284 Received 4th October
NUCLEAR EXPRESSION OF AN ENVIRONMENTALLY FRIENDLY SYNTHETIC PROTEIN BASED POLYMER GENE IN TOBACCO CELLS X. Zhang I, C. Guda j, R. Datta t, R. Dute I, D. W. Urry 2 and H. Danieli t* IMolecular Genetics Program, Department of Botany and Microbiology, 101, Life Sciences Building, Auburn University, Auburn, AL 36849-5407 2Laboratory of Molecular Biophysics, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294-0019 SUMMARY We report here expression of a protein based polymer gene (Gly-Val-Gly-Val-Pro)m , coding for three amino acids in a pentamer sequence repeated 121 times via the nuclear' ge~ome of tobacco cells. Transformed tobacco cells were obtained by particle bombardment. Stably transformed cells show the presence of the polymer gene in the tobacco nuclear genome (2-5 copies); introduced polymer gene is transcribed efficiently as revealed by Northern blots; Western blots show the presence of the polymer protein. To the best of our knowledge, this report represents the first demonstration of expression of a synthetic gene (with no natural analog) in higher plants.
INTRODUCTION Elastic and plastic protein-based polymers (or bioelastie materials), defined as high polymers of repeating peptide sequences, offer a range of materials similar to that of oil-based polymers, such as hydrogels, elastomers and plastics. Protein-based polymers have their origins in repeating sequences that occur in all sequenced mammalian elastin proteins (Yeh et al., 1987). In the most striking examples, the sequence (Vall-Pro2-Gly3-Val4-Gly5)n occurs in bovine elastin with n=l l, without a single substitution (Yeh et al., 1987). Protein-based polymers can be prepared of varied design and composition and can be made biodegradable with chemical clocks to set their half lives (Urry, 1995). Protein-based polymers tested to date have been shown to have remarkable biocompatibility, thereby enabling a whole range of medical applications including the prevention of post-surgical adhesions, tissue reconstruction and programmed drug delivery (Urry et al., 1993). For example, the polymer poly(Gly-Val-Gly-Val-Pro), used in this study, has been shown to prevent adhesions in the rat contaminated peritoneal model following abdominal injury (Urry el al., 1993). On the non-
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medical side, there are transducers, molecular machines, superabsorbant agents, biodegradable plastics, and controlled release of agricultural crop enhancement agents, such as herbicides, pesticides, growth factors and fertilizers (Urry et al., 1993). What is required for the commercial viability of such protein-based polymers is a cost of production that would begin to rival tlmt of oil-based polymers. The potential to do so resides in tow cost bioproduction. So far several protein-based polymers have been produced in E. coli through genetic engineering (McPherson et al., 1992; Krejchi et al., 1994; Urry et al., 1994: Guda et al., 1995; Daniell et al., 1995a). In our previous study, a synthetic polymer gene, coding for (Gly-Val-Gly-Val-Pro)120, was hyper expressed in E. coli to the extent that polymer inclusion bodies occupied nearly 80-90% of the cell volume (Guda et al., 1995; Daniell et al., 1995a; Urry et al., 1994). llowever, current production through fermentation is still an expensive process. A possible strategy tbr reducing the production cost would be to produce polymers in plants. Therefore, in this study we investigate the potential for expression of a protein-based polymer gene, coding for (Gly-Val-Gly-VaI-Pro)121, in tobacco cells through the nuclear genome.
MATERIALS AND METHODS Plant materials and bombardment conditions Suspension cultures of Nicotiana tabacum L,, line NTI were maintained as described (Daniell, 1993). Bombardment of tobacco cells and post-bombardment selection of stably transformed cell lines were performed according to Daniell (1993, 1995). Southern blot analyses Total DNA was isolated from suspension cells essentially as described by Rogers and Fraley (1988). Total DNA (10 I-tg) was digested with Band-tl or EcoRI, electrophoresed through 0.7% agarose gels, and transferred to MSI membranes (Micron Separation Inc. Westboro, MA). Prehybridization and hybridiz~ation were done according to Daniell et al. (1995b). Radiolabelled DNA probe (1.8 kbp) was prepared by the random-primed labeling procedure (Promega). After washing, blots were exposed to Fuji X-ray film at -80 ° C with two intensifying screens.
Northern blot analyses Total RNA was isolated from suspension cells essentially as described by Vries et al. (1988). RNA (20 lLtg)was denatured by formaldehyde treatment, separated in a 1.2% agarose gel in the presence of formaldehyde, and transferred to a MSI membrane. The blot was prehybridized and hybridized as described above for Southern blot analysis. Western blot analyses Plant cells were homogenized by grinding in liquid N 2. SDS-PAGE buffer (62.5 mM Tris-HCI, pll 6.8, 3% SDS, 25% glycerol, 5% mercaptoethauol, I mM phenylmethylsulfonyl fluoride) was then added. Samples were boiled for 5 rain. Insoluble debris was removed by
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centrifugation at 12,000 g. Proteins (15 ~tg) were separated by SDS-PAGE according to Laemmli (1970), transferred to a nitrocellulose membrane and stained with antiserum raised against the polymer AVGVP (provided by the university of Birmingham, monoclonal facility). Total protein contents were determined by the method of Bradford (Bradford, 1976) with Bovine Serum Albumin as a standard. RESULTS AND DISCUSSION The protein-based polymer gene coding for three amino acids glyeine, valine and proline, (GVGVP)m, has been described in detail elsewhere (Daniell et al., 1995a). The nuclear vector for stable expression of the 121mer polymer protein was constructed as follows. The uidA gene was removed from the plasmid pBI121 (Jefferson, 1987) as a XbaI-SstI fragment and replaced by the 121 mer polymer fragment (obtained as XbaI-SstI fragment from pUC118 plasmid) resulting in the construct pBI121-XZ-120mer (Fig. I). /HindlII /~SphI stI~l~~baI amHI
I12
\
vo. . . . /
~
/HindIII \KpnI SstI
Figure 1. The plasmid pBI121-XZ-120mer. Abbreviations used in the plasmid map are: RB, T-DNA right border; nos-pro, nopline synthase gene promoter; NPTII, neomycin phosphotransferase gene that serves as a selectable marker; nos-ter, nopline synthase gene terminator; CaMV 35S, CaMV 35S promoter; EGl20mer, coding sequence for (GVGVP)I2t; LB, T-DNA left border.
After bombardment, three kanamycin-resistant cell lines were recovered. They were maintained in the growth medium containing kanamycin (100 I.tg/ml) and subcultured once a week. To determine if the polymer gene was stably integrated into the nuclear genome of tobacco cells, Southern analysis was performed on kanamycin-resistant cells which had been maintained and subcultured in the growth medium for two months (Fig. 2A). Two out of three kanamyeinresistant clones (clone #3 and #8) tested showed a hybridizing band of the expected size (1.8 kb, when the nuclear DNA was digested with BamHI, Fig. 2A, lanes 2,3), whereas lane 4 (Fig. 2A) containing DNA from untransformed cells lacked this band. Southern analysis did not detect hybridizing bands other than expected 1.8 kb restriction fragment, indicating the specificity of the probe; this also indicates that transformants contain only intact copies of the polymer gene in
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the tobacco nuclear genome, The number o f intact polymer gene copies was estimated to be two to five. The copy number reconstruction (Fig. 2A, lanes 5, 6, 7; 1, 2, 5 copies) was based upon a tobacco nuclear genome size of 4.8
x 10 9
bp. Southern blot of EcoRI-digested genomie DNA
from transformed cells showed a hybridizing fragment of a larger size for these two clones (data not shown).
Figure 2. Southern, :Northern, and Western blot analyses of control and transgenic tobacco NT1 cell lines. (A) Southern blot of BamHl digested genomic DNA from control (lane 4) and three (lanes 1,2, and 3) kanamycin resistant cell lines (transgenic lines 1, 3, and 8 respectively). Lanes 5, 6, and 7 contain standards equivalent to 1, 5, and 25 copies of the polymer gene, respectively. Molecular weight markers are shown on the left. (B) Northern blot of RNA isolated from control (lane 1) and transgenic tobacco cell line 3 (lane 2) and celt line 8 (lane 3). (C) Western blot using the poly (AVGVP) antibody to probe purified polymer proteins from E. coli (lane 1) and crude protein extracts of control (lane 2) and transgenic tobacco cell line 3 (lane 3). Northern analysis was performed to determine if the polymer gene was transcribed in stably transformed cells which had been maintained and subcultured in the growth medium for more than two months. Figure 2B clearly shows a hybridizing transcript of the expected size (1.8 kb) in transformant #3 (lane 2), but not in control (lane 1) containing RNA from untransformed cells. Polymer transcript was not detected in lane 3 containing RNA from transformant #8. In addition, a smaller transcript was detected in transformant #3. The origin of a smaller transcript
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remains unclear. It could be the product of incomplete transcription or in vivo degradation product of the 1.8 kbp transcript that accumulated to detectable levels. Smaller uidA transcripts have been observed in stably transformed sorghum cell cultures obtained by particle bombardment in a previous study (Hagio et al., 1991). Western analysis was performed to detect the presence of the polymer protein in stable transformant #3 (Fig. 2C), which showed a strong transcription signal in the Northern blot. The Western blot detected the polymer protein in the transformed clone #3 (Fig. 2C, lane 3), whereas the polymer protein was not detected in control untransformed tobacco ceils (Fig. 2C, lane 2). Polymer production was also confirmed by ELISA for transformed clone #3 (data not shown). Although, the polymer protein was accumulated to a detectable level by Western blot analysis, the amount of the polymer protein is much less than expected from the level of mRNA detected. The reasons for lesser accumulation of the polymer protein are unclear. A possible explanation could lie in the polymer coding sequence, which has a prokaryote-preferred codon composition. It is well known that foreign genes are expressed at different levels in various cellular compartments based on codon preference and usage (Perlak, 1991). For example, a major hurdle in engineering insect resistant plants has been the low level of expression of the Bacillus thuringiensis (B. t.) toxin gene; increase in B. t. toxin gene expression of up to 500-fold has been achieved through specific modification of the B. t. coding sequence to suit the eukaryotic nature of plant nuclei (Perlak, 1991). Therefore, a parallel
nuclear expression study using the EG-
130mer gene with the tobacco nuclear-preferred codons is currently being conducted in our laboratory. Additionally, availability of amino acids could also be a limiting factor for high level expression of this polymer. In photosynthetic tissues, glycine is formed by transamination of glyoxylate, which is generated from glycolate produced from Rubisco as a consequence of photorespiration (Atkins and Beervers,
1990). In non-photosynthetic tissues, such as
heterotrophic NTI tobacco cells used in our study, the supply of glycine would be much reduced due to the absence of Rubisco-generated glycolate (Atkins and Beervers, 1970); therefore, translation of polymer transcripts could be minimal in heterotrophic tobacco cells because of limited availability of glycine which accounts for 40% of the total amino acid pool required for synthesis of the polymer protein (GVGVP),,o. Further studies are therefore needed to address this question; polymer gene may be expressed in autotrophic tobacco cells, and in addition, the transformed cells may be grown in the presence of the three amino acids (glycine, valine, and proline) required for maximum synthesis of polymer. These experiments are currently in progress and should provide essential information on the amino acid pools required for enhanced polymer production.
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ACKNOWLEDGMENTS
This work was supported in part by: the USDA grants # 93-37311 and 95-02770 and the N1H grant # GM 16551-01 to HD and the Office of Naval Research grant # N0014-89-JI970 to DWU.
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