1. Cloning And Expression Of Human α-synuclein CDNA 2. Heterologous Expression Of Chloroperoxidase In P.pastoris System

α-Synuclein is present primarily in presynaptic terminals in the central nervous system. More attention has been paid to the protein, because of the discovery of a relationship between its dysfunction and several neurodegenerative diseases, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). The physiological functions ofα-synuclein remain to be fully defined. Some reports showed thatα-synuclein was involved in the regulating membrane stability and neuronal plasticity, and anti-apoptois of neurons. It also showed the chaperone-like activity and took part in the signal transduction. Other results demonstrated that its abnormal accumulation and aggregation would lead to theα-synucleinopathies, including PD. This process is a cascade of events involving misfolding or loss of normal function ofα-synuclein triggered by various factors, either environmental or genetic. At higher concentrationsα-synuclein (including its mutated forms A53T and A30P) has a propensity to self-aggregate, leading to the formation ofα-synuclein oligomers (protofibrils, fibrils) and polymers, but evidences has showed that the protofibrillization rate of both mutants is higher than that of wild-tyPe protein; and the fibrillization rate is lower in A30P and higher in A53T. The oligomers (protofibril and fibril) ofα-synuclein are implicated to be toxic to neurons and result in the cell apoptosis. Asα-synuclein has such pathological properties and the formation of intraneuronalα-synuclein aggregates is characteristic of several neurodegenerative diseases, including Parkinson's disease (PD), it may be used as a potential target for the development of novel anti-PD drugs.In this paper, the cDNA of humanα-synuclein was successfully cloned. A recombinant transgenic plasmids carrying theα-synuclein coding region was partially construted, which will be used to prepare the transgenic DNA fragment for the construction of transgenic rat.α-synuciein cDNA was also transformed into Pichia pastoris system.Total RNA was extracted from the human SH-SY5Y cells induced by dopamine.α-Synuclein cDNA was obtained by RT-PCR, with the total RNA as a template. DNA sequencing .and BLAST showed that the cDNA was homologous to the reported sequence with 100% identity (GenBank: BC₀13293). The A53T mutation ofα-synuclein was achieved by site-directed mutation method. Other procedures are as follows:1) The A53T mutant gene was subcloned into pcDNA3.0 witch contains BGH polyA site. A piece of DNA fragment harboring the A53T coding sequence and the BGH polyA site was obtained by PCR. The recombinant expression plasmid for the transgenic rat was partly constructed.2) The A53T mutant cDNA was inserted downstream ofα-signal peptite encoding gene in the plasmid pPIC9K, forming the recombinant plasmid pPIC9K-Sym. The recombinant plasmid pPIC9K-SyGFP carrying the synuclein-GFP fusion gene was also constructed. After linearized by the restriction enzyme Sac I, the recombinant plasmid pPIC9K-Sym and pPIC9K-SyGFP were transformed into P. pastoris GS115 respectively, by electroporation. The His⁺Mut⁺ transformants were screened using MD medium and G418. After identified by PCR, the transformants with correct phenotype were fermented with BMMYC medium, induced by 1% methanol. The filtrate of culture was analyzed using SDS-PAGE, and the result showed that the distinctive bands, with a molecular weight of approx. 40kDa or 20kDa, were found respectively in the transformants GS115/pPIC9K-SyGFP and GS115/pPIC9K-Sym, but not in the control at the corresponding position. Western blot analysis showed that they were recombinant humanα-synuclein.3) The A53T mutant cDNA was also inserted in the intracellular expression plasmid pPIC3.5K, and the recombinant plasmids pPIC3.5K-Sym and pPIC3.5K-SyGFP were constructed as mentioned in 2). After linearized by the restriction enzyme Sac I, the recombinant plasmid pPIC3.5K-Sym and pPIC3.5K-SyGFP were transformed into P. pastoris GS115 respectively, by electroporation. Multi-copy transformants were screened using high concentration G418 plate. After fermentation in BMMYC medium, induced by 1% methanol, the strong fluorescence in yeast cells was observed by fluorescence microscopy and it was implied that abundant fusion protein was expressed in the host cells. Transformants cultured in liquid or solid medium show improving survival ability instead of apoptosis, probably due to the absence of the proper environmental and genetic conditions. UV mutagenesis was made on the transformant GS115/pPIC3.5K-SyGFP and severalα-synuclein sensitive mutants, including Uv13, were acquired. Preliminary result.exhibited that the mutant Uv13 grew much more slowly in MM medium (carbon source: MeOH) than in MD medium (carbon source: Glucose). The growth rate of the mutant was also much lower than that of the control transformant in the same MM medium. Optimization of this model and using it in high-throughput screening would facilitate the search for molecules that alleviateα-synuclein-induced cytotoxicity. Chloroperoxidase (CPO1; EC 1.11.1.10) is secreted by the filamentous fungus Caldariomyces fumago and is a heavily glycosylated monomeric hemoprotein, with a broad spectrum of oxidative characteristics. The molecular weight of CPO is approximately 42 kDa. It shares features of both classical peroxidases and cytochrome P450s. In addition to catalyzing halogenation, CPO catalyzes a variety of synthetically enantioselective oxygen transfer reactions. Many useful compounds were produced by CPO catalyzing on asymmetric epoxidation which could be used as materials in fine chemicals and enantiomerically pure intermediates, Besides its catalytic asymmetric epoxidation of olefins, CPO catalyzes carbon-hydrogen bond hydroxylation, sulfoxidation, enantioselective oxygen and kinetic resolution in alcohol with high catalytic yield and turnover number in comparison with other peroxidases. But its enzyme activity is limited by the structure of the substrate during asymmetric epoxidation. Another limit is that the optimal growth temperature of the fungus is 19℃, and at this temperature it grows slowly. Gene engineering method is a good choice for gettiing abundant and stable CPO enzyme. For using site-directed mutagenesis and other molecular evolution method to explore the structure-function of CPO and modify the enzyme protein, an efficient expression system for cpo gene is required. At present, many attempts at heterogenous expression of CPO were unsuccessful. Some scientists supposed that its mRNA was unstable during in the recombinant expression. In this paper, we tried to find the possible reasons that affect CPO heterologous expression, and attempted to express CPO in P. pastoris at a high level. The cpo gene was successfully cloned from C. furnago, DNA sequencing and BLAST showed that the gene was homologous to the reported sequence with 100% identity (GenBank: AJ₃00448). Several constructs were made which carried the cpo/gfp fusion gene inserted downstream of theα-signal peptite encoding sequence. After linearized by the restriction enzyme Sac I, the recombinant plasmids pPICgK-CPO, pPICgK-CKG, pPICgK-GC, pPIC9K-GKC and pPIC9K-GFP were transformed into P. pastoris GS115, respectively, by electroporation. The His+Mut+ transformants were screened using MD medium and G418. After identified by PCR, the transformants with correct phenotype were cultivated in BMMYC medium, induced by 1% methanol. The cultural filtrate was analyzed by SDS-PAGE, and the result showed that a distinctive band, with a molecular weight of approx.30kDa was found in the transformant GS 115/pPIC9K-CKG but not in the control at the corresponding position. The RFU of GFP in the supematant of GS115/pPIC9K-CKG was markedly high Compared to that of the control, suggesting that the CPO gene was expressed in P. pastoris. As the CPO gene was fused upstream of the GFP gene, it is reasonable to consider that the mRNA is stable in P. pastoris expression system. However, no activity was detected in any transformant supernatant, implying that the CPO protein is probably unstable. The stable and active recombinant protein might be obtained by DNA shuffling or other molecular methods.