Secretory Expression Of SOD In S.cerevisiae

Background and ObjectiveSuperoxide dismutase (SOD) is a metal-containing enzyme which can be found abundantly in many organisms, from microorganisms to plants and animals. There are three types of SOD, classified according to the metal ion present at the enzyme active site: Cu/Zn-SOD, Mn-SOD and Fe-SOD. The SOD family catalyzes the dismutation of superoxide radicals into hydrogen peroxide and oxygen. Therefore, it can guard against oxygen poison, strengthen resistance to radiation damage and delay senility. It has good cure effects in some tumors, inflammatory diseases and self-immunity diseases. Among these, Cu, Zn superoxide dismutase (SOD1) is widely distributed and comprises 90% of the total SOD. This ubiquitous enzyme has great physiological significance and therapeutic potential in several diseases, such as familial amyotrophic lateral sclerosis(FALS), Parkinson's disease, Alzheimer's disease, dengue fever, cancer, Down's syndrome,cataract, and several neurological disorders.The native SOD are limited and appear variant protain immunogenic could not be accepted by human. The exploration of the therapeutic potential of SOD has been hindered by its limited availability.For this reason it would be preferable to use a recombinant human SOD that is identical to authentic enzyme.At present, a great deal of research work has been done to its foundation and clinical practice in overseas. But in our country the research work always focus on the basic theoretics related application. The reports of SOD clone and expression are less. Human SOD was traditionally cloned and expressed in E.coli, so it's very hard to separation and purification. In this report an gene coding forhuman Cu/Zn-SOD was synthesized and cloned into a yeast expression and secretion vector containing the mating factor a pre-pro leader sequence. This simple approach offers the production for a potential practical application in cosmetic and eatable and medical treatment field. Thus it could exert the function as to against oxygen poison and delay senility. The study lay a good basis of large scale purification and further practical research ofhCu/Zn-SOD.Methods1. PCR amplification of Mating factor -a secretion signal cascade: The MF- a genes were amplified from the S. cerevisiae INVScl genomic DNA as a template. The amplification primers were designed to provide Kpn I and BamH I restriction sites.2. PCR amplification of hCu/Zn-SOD: The total RNA was extracted from fresh human stomach by using Rneasy Minikit and amplification of cDN A was performed by using the CAS super TWO-STEP RT-PCR kit. the cDNA encoding human Cu/Zn-SOD was amplified by RT-PCR using the total cDNA as the template. The amplification primers were designed to provide BamH I and Xbal restriction sites.3. Construction of the plasmid pUC-MS: the human Cu/Zn-SOD cDNA with the yeast mating factor(MF) a signal Sequence was constructe into vector pUC19 resulting in recombinant plasmid pUC-MS, and then we identified the new plasmid.4. Construction of the plasmid pETSOD: The correct recombinants pUC-MS confirmed by DNA sequencing were digested with BamHI and Hindlll before being ligated into the corresponding restriction sites of the expression vector pET22b(+) under T7 promotor. The final constructed plasmid pETSOD was transformed into the bacterial strain BL21(DE3)pLysS.5. Construction of the plasmid pYES-MS: The correct recombinants pUC-MS were digested with Kpnl and Xbal before being ligated into the corresponding restriction sites of an E.coli/ S.cerevisiae shuttle vector pYES2 resulting in recombinant expression plasmid pYES-MS which was then thansformed into Saccharomyces cerevisiae INVScl.6. Overexpresstion of human Cu/Zn-SOD in E. coli: The recombinant enzyme was overexpressed in E. coli cells by using the strong inducible T7 promoter under control of the presence of isopropyl-b-thiogalactopyranoside(IPTG).Then we analysised the total protein by 15% SDS-PAGE and western-blot. SOD activity was assayed by the NBT photoredction method.7. Overexpresstion of human Cu/Zn-SOD in S.cerevisiae: Yeast strains weretransformed by the lithium acetate method. Transformants were re-streaked to SC-ura, and single colonies selected for use in further experiments. The transformant pYES-MS/INV was cultured in YPD culture medium by induction with 2% galactose. Cells were harvested from 1 1 cultures bycentrifugation ,then disrupted with a glass homogenizer. The culture medium supernant were concentrated by Mini-filter.Then we analysised the total protein and supernant protein extract by 15% SDS-PAGE and western-blot. SOD activities were visualized in non-denaturing polyacrylamide gels by the inhibition of NBT dye reduction.Results1. The MF- a genes were amplified from the S. cerevisiae INVScl genomic DNA as a template.The sequencing result shows that the inset is composed of 250bp. From the total RNA of human stomach, hCu/Zn-SOD cDNA as a PCR product with length of 0.47kb as judged by agarose gel electrophoresis was amplified using RT-PCR kit. The ORF is 462bp long and ends with the stop codon TGA.2.The coding sequence of human Cu/Zn-SOD was subcloned into the expression vector pET22b(+) between BamHI and Hindlll sites. Once the correct recombinants pETSOD were further confirmed by double restriction enzyme digestion, the 460bp insert sequence would be found by agarose gel electrophoresis.3. The correct recombinants pUC-MS confirmed by DNA sequencing were digested with Kpnl and Xbal .The correct recombinants pYES-MS were further confirmed by double restriction enzyme digestion, the 720bp insert sequence would be found by agarose gel electrophoresis.4. The final constructed plasmid pETSOD was transformed into the bacterial strain BL21(DE3)pLysS. The recombinant enzyme was overexpressed in E. coli cells under control of the presence of IPTG. SDS-PAGE and Western-blot analysis revealed that the recombinant E. coli expressed the predicted 16kDa human Cu/Zn-SOD, and its amount was over 20% of total proteins of the the bacteria in soluble form, which had specific SOD activity.5. Yeast cells transformed with pYES-MS were induced with 2% galactose. After the recombinant gene expression, both extra- and intracellular hCu/Zn-SOD were analyzed by SDS-PAGE and Western blot. The culture medium was centrifuged at room temperature, the cell-free supernatant extract was mixed with lOul of 2x SDS sample buffer, and the cell pellet was resuspended in an equal volume of 1 x SDS sample buffer. SDS-PAGE analysisrevealed that the recombinant Yeast cells expressed the predicted 21kDa intracellular hCu/Zn-SOD and 19kDa extracellular hCu/Zn-SOD .The intracellular amount was over 10% of total proteins. Immunoreactivities of both extra- and intracellular hCu/Zn-SOD were analyzed by Western blot.The secretion level of hCu/Zn-SOD was considerably higher than the recombinant which non-induced with galactose. Both extra- and intracellular hCu/Zn-SOD extracts from pYES-MS/ INV induced contained considerably more SOD activity than did matrix from pYES-MS/ INV non-induced.ConclusionIn our experiment, we have successfully used the yeast mating factor(MF) a signal Sequence as a fusion expression partner for the production of hCu/Zn-SOD which has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule. At mean time we constructed plasmid pETSOD was transformed into the bacterial strain BL21(DE3)pLysS. The enzyme was overexpressed in E. coli cells under control of the presence of IPTG. SDS-PAGE and Western-blot analysis revealed that the recombinant E. coli and S. cerevisiae expressed the predicted human Cu/Zn-SOD, Immunoreactivities of both extra- and intracellular hCu/Zn-SOD were analyzed by Western blot. The hCu/Zn-SOD biologically activities have been visualized in non-denaturing polyacrylamide gels by the inhibition of NBT dye reduction. The study lay a good basis of large scale purification and further practical research of human Cu/Zn-SOD .