Escherichia coli is the “workhorse” for the production of recombinant proteins due to numerous advantages. It is well known for its ease of handling with short time generation and for its ability to accumulate high amount of foreign protein. Nevertheless E. coli systems present also some limits; serious problems can occur during the process of heterologous gene expression and purification, as low expression rates, formation of inclusion bodies, improper protein-folding and inability to produce complex disulfide bonds. This is due to lacks fundamental prerequisites for efficient secretion, due to the membrane structure, the low chaperone and foldase level and the high periplasmatic protease concentration. Alternative expression hosts are the Gram-positive Bacillus strains, particularly B.subtilis and B.megaterium, designated as GRAS (generally recognized as safe) organisms, free of any endotoxin. Furthermore, compared with E.coli, B.subtilis and B.megaterium offer high biosynthetic capacity and an efficient secretion apparatus that guides the expressed proteins directly into the culture supernatant. The aim of this project was to evaluate these microorganisms as expression hosts 1) either for production of heterologous eukaryotic proteins, as the anti-prion 8H4 antibody fragment (cap.I-III), 2) either for engineering of a natural metabolic pathway, as biosynthesis of hyaluronic acid (cap.IV). 8H4 scFv (Single chain Fragment variable) is an eukaryotic complex molecule with three disulphide bonds, that has been recently used as therapeutic approach in prion diseases, inhibiting prion replication and delaying the development of prion disease. In order to optimize this therapeutic perspective and address the 8H4 scFv to its targets in SCN, 8H4 scFv has been cloned in fusion with peptide-transduction domain of HIV-1 TAT protein, that penetrates efficiently into cells translocating across the plasma membrane and also the blood-brain barrier. TAT 8H4svFv protein was been produced previously in our laboratory in E.coli, but with very low yields. In this study, WB800N B.subtilis (deleted of 8 extracellular proteases) and MS941 B.megaterium were engineered to produce and secrete anti-prion TAT-8H4scF protein . Several expression-secretion vectors have been constructed, cloning the wild-type or a synthetic E. coli optimized TAT 8H4 scFv sequence, under the control of different promoters (IPTG-inducible Pgrac or strong constitutive P43 promoters in B.subtilis, or xylose-inducible T7 promoter in B.megaterium), in fusion with secretion signal peptide AmyQ. Although the chosen Bacillus strains were deleted of several extracellular proteases, no clearly TAT 8H4scFv secreted was revealed in our hand, (neither after IMAC purification of culture medium, neither after ammonium sulphate precipitation). Antibody was present overall in insoluble intracellular fraction, and a small fraction was revealed in periplasm. (cap.II) Because the high secretory capacity of Bacillus strains is not appeared, we have tried to take advantage from the use of fusion protein technology to increase the yields and solubility of TAT8H4scFv in E.coli cytoplasm. At the aim bacterial chaperone DnaK and like-chaperone α -synuclein protein were been chosen as fusion tags, for their activity favouring refolding together physic-chemical characteristics. Although fusions of TAT 8H4scFv to α -synuclein and DnaK increase expression resulting in accumulation of significant levels of antibody, these fusion proteins show to be largely insoluble. Thus, we have tried to co-express DnaKJE and GroELS bacterial chaperones together α-syn or DnaK fusion-8H4scFvs, and purify α-synTAT8H4 from more oxidizing periplasmic environment. Since the fusion-scFvs yields in soluble form remained not significant, we have purified Dnak TAT8H4 and α -syn TAT8H4scFvs in denaturing conditions by immobilized metal affinity chromatography (IMAC) and in vitro refolding. In this way has been possible to obtain correctly refolded α-syn 8H4, that is able to recognize human prion protein by immunoblotting. When Dnak TAT8H4 and α -syn TAT8H4 were added to CHO cells culture medium, they were rapidly delivered inside the cells, and displayed mainly a nuclear localization. α-syn TAT8H4 antibody fragment is able to deplete the superficial membrane bound prion protein in HeLa transfected cells with plasmids GFP-PrP but not the analog GFP-Doppel. Time course of these protein revealed that α -syn TAT8H4 shows a half-life major than Dnak TAT8H4 (cap.III). In conclusion fusion protein technology is revealed effective to increase the yields of TAT8H4scFv in E.coli cytoplasm α-synuclein is demonstrated preferable to DnaK in fusion with TAT8H4 scFv, either for lower molecular weight in fusion protein, than for higher yields and ability to refold. Due to its specificity to deplete PrP, α-syn TAT8H4 scFv could be effective in spongiform transmissible diseases treatment. Moreover, the use of cell-permeable antibodies, due to TAT transduction domain, would avoid the safety and ethical concerns surrounding the direct application of recombinant DNA technology in human clinical therapy and could be extended to treatment of other pathology. The second part of this study concerns the engineering of a natural metabolic pathway, as the biosynthesis of hyaluronic acid HA. In our project the HA biosynthesis pathway present in natural producers as streptococci has been has been adapt to Bacillus. Genes involved in the pathway of synthesis for the precursor sugars, that are disperse in the genoma of Bacillus subtilis , were here linked in a unique polycistronic mRNA togheter with hyaluronan synthase from Streptococcus equi, as is present in natural pathway of streptococci. In our case we have developed several episomal vector where several genes are under the control of strong and inducible promoters. This expression system, using of a plasmid with relatively higher copies, it has advantage to express more higher levels of mRNA than integrative system on the chromosome. By PCR amplification of hasA and tuaD genes from S.zooepidemicus and B.subtilis respectively, and cloning in pHT B.subtilis/E.coli shuttle vector under Pgrac, we have selected stable metabolic engineered 1012 and WB800N Bacillus subtilis strains, secreting HA with molecular weights higher of 800 kDa of Streptococcus with a yield more than 5g/L, that are actually produced by Strepctococcus in industrial HA and largely exceeding that is published. To further increase yields and HA molecular weights, we have construct and cloned the cassette-operons hasA-tuaD and hasA-tuaD-gtaB-pgi under inducible promoter T7 in B. megaterium. Althought T7 expression system is present also in E.coli , we have demonstrated that recombinant E.coli cells produce only low amounts of HA. Indeed these engineered B.megaterium strains in the optimal expression conditions identified produce about 2g/L in shake flask, that are very promising results in view of batch fermentation cultures. Moreover hasA-tuaD-gtaB-pgi overexpressing B.megaterium seem produce higher HA MW, about 1800 kDa, comparable also for polydispersity to commercially available Streptococcus, suggesting that gtaB-gpi overexpression result in a molecular weights enhancement (cap.IV). In conclusion, although high potential secretory capacity of Bacillus was not appear in secretion of a heterologous protein, as anti-prion 8H4 scFv, B. subtilis and B.megaterium have proven to be superior expression hosts for engineering of a natural metabolic pathway, as biosynthesis of HA, based on several criteria: 1) good quality of HA, comparable to commercial streptococcus standards regards to molecular mass and polydispersity, and superior regards yields. 2) In addition, unlike Streptococcus, the B. subtilis and B.megaterium-derived HA products are exotoxin free and secreted directly into the surrounding medium and are not cells associate, simplifying the recovery process. 3) Finally, while Streptococcus A and C require more expensive complex media for growth, Bacillus strains grow on minimal media, assuring a final products more pure and toxin-free

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

Synuclein is a soluble, natively unfolded protein that is highly enriched in the presynaptic terminals of neurons in the central nervous system. Interest in -synuclein has increased markedly following the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinson's disease. The physiological functions of -synuclein remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. In addition, there is increasing evidence pointing to phosphorylation as playing an important role in the oligomerization, fibrillogenesis, Lewy body formation, and neurotoxicity of -syncline in Parkinson's disease. Immunohistochemical and biochemical studies reveal that the majority of -synuclein within inclusions from patients with Parkinson's disease and other synucleinopathies is phosphorylated at Ser129. -Synuclein can be phosphorylated in vitro also at Ser87, and three C-terminal tyrosine residues (Tyr125, Tyr 133, and Tyr136). Tyrosine 125 phosphorylation diminishes during the normal aging process in both humans and flies. Notably, cortical tissue from patients with Parkinson's disease-related synucleinopathy dementia with Lewy bodies showed less phosphorylation at Tyr125. While phosphorylation at Ser87 is enhanced in synucleinopathies, it inhibits -synuclein oligomerization, and influences synuclein-membrane interactions. The possibility that -synuclein neurotoxicity in Parkinson's disease and related synucleinopathies may result from an imbalance between the detrimental, oligomer-promoting effect of Ser129 phosphorylation and a neuroprotective action of Ser87/Tyr125 phosphorylation that inhibits toxic oligomer formation merits consideration, as will be discussed in this article.