Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants.

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.

Microsatellite analysis and marker development in garlic: distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae.

Allium vegetables, such as garlic and onion, have understudied genomes and limited molecular resources, hindering advances in genetic research and breeding of these species. In this study, we characterized and compared the simple sequence repeats (SSR) landscape in the transcriptomes of garlic and related Allium (A. cepa, A. fistulosum, and A. tuberosum) and non-Allium monocot species. In addition, 110 SSR markers were developed from garlic ESTs, and they were characterized-along with 112 previously developed SSRs-at various levels, including transferability across Alliaceae species, and their usefulness for genetic diversity analysis. Among the Allium species analyzed, garlic ESTs had the highest overall SSR density, the lowest frequency of trinucleotides, and the highest of di- and tetranucleotides. When compared to more distantly related monocots, outside the Asparagales order, it was evident that ESTs of Allium species shared major commonalities with regards to SSR density, frequency distribution, sequence motifs, and GC content. A significant fraction of the SSR markers were successfully transferred across Allium species, including crops for which no SSR markers have been developed yet, such as leek, shallot, chives, and elephant garlic. Diversity analysis of garlic cultivars with selected SSRs revealed 36 alleles, with 2-5 alleles/locus, and PIC = 0.38. Cluster analysis grouped the accessions according to their flowering behavior, botanical variety, and ecophysiological characteristics. Results from this study contribute to the characterization of Allium transcriptomes. The new SSR markers developed, along with the data from the polymorphism and transferability analyses, will aid in assisting genetic research and breeding in garlic and other Allium.

Translational fusion and redirection to thylakoid lumen as strategies to enhance accumulation of human papillomavirus E7 antigen in tobacco chloroplasts.

Human papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death in women worldwide, and its E7 antigen is the major candidate for a therapeutic vaccine. The large scale production of E7 by molecular farming that would lead to the development of a safe and inexpensive vaccine is impaired by its low accumulation level in the plant cell. To enhance antigen production in the plastids, two alternative strategies were carried out: the expression of E7 as a translational fusion to ß-glucuronidase enzyme and redirection of E7 into the thylakoid lumen. The use of the ß-glucuronidase as a partner protein turned out to be a successful strategy, antigen expression levels were enhanced between 30 and 40 times relative to unfused E7. Moreover, best accumulation, albeit at a high metabolic cost that compromised biomass production, was obtained redirecting E7 into the thylakoid lumen by the incorporation of the N-terminal transit peptide, Str. Following this approach lumenal E7 production exceeded the stromal by two orders of magnitude. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts.

Transformation of Solanum tuberosum plastids allows high expression levels of ß-glucuronidase both in leaves and microtubers developed in vitro.

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of ß-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of ß-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on ß-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased ß-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants.

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135-160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the beta-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.

Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast.

Cervical cancer linked to infection with human papillomavirus (HPV) is the third cause of cancer-related death in women. As the virus cannot be propagated in culture, vaccines have been based on recombinant antigens with inherited high-cost production. In a search of alternative cheap production system, E7 HPV type 16 protein, an attractive candidate for anticancer vaccine development, was engineered to be expressed in tobacco chloroplast. In addition, E7 coding sequence was fused to potato virus X coat protein (CP) to compare expression level. Results show that E7CP transcript accumulation reached lower levels than non-fused E7. However, antigen expression levels were higher for fusion protein indicating that CP stabilizes E7 peptide in the chloroplast stroma. These results support viability of transplastomic plants for antigen production and the relevance of improving recombinant peptide stability for certain transgenes to enhance protein accumulation in this organelle.

Sperm protein "DE" mediates gamete fusion through an evolutionarily conserved site of the CRISP family.

The first member of the cysteine-rich secretory protein (CRISP) family was described by our laboratory in the rat epididymis, and it is known as DE or CRISP-1. Since then, numerous CRISPs exhibiting a high amino acid sequence similarity have been identified in animals, plants and fungi, although their functions remain largely unknown. CRISP-1 proteins are candidates to mediate gamete fusion in the rat, mouse and human through their binding to complementary sites on the egg surface. To elucidate the molecular mechanisms underlying CRISP-1 function, in the present work, deletion mutants of protein DE were generated and examined for their ability to bind to the rat egg and interfere with gamete fusion. Results revealed that the egg-binding ability of DE resides within a 45-amino acid N-terminal region containing the two motifs of the CRISP family named Signature 1 and Signature 2. Subsequent assays using synthetic peptides and other CRISPs support that the egg-binding site of DE falls in the 12-amino-acid region corresponding to Signature 2. The interesting finding that the binding site of DE resides in an evolutionarily conserved region of the molecule provides novel information on the molecular mechanisms underlying CRISP-1 function in gamete fusion with important implications on the structure-function relationship of other members of the widely distributed CRISP family.

Expression and structure-function analysis of de, a sperm cysteine-rich secretory protein that mediates gamete fusion.

Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.