Clone And Expression Of Acetyl CoA Carboxylase CT Domain

Acetyl-CoA carboxylases are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases. The carboxyltransferase (CT)domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim. People have determined the crystal structures of the CT domain of yeast ACCase in complex with the herbicide haloxyfop or diclofop. The inhibitors are bound in the active site, at the interface of the dimer of the CT domain.ACCases have been found in most living organisms. There are mainly two kinds of ACCases, including procaryotic enzyme and eukaryotic enzyme. Multi-subunit ACCase, while multi-domain ACCase mainly in humans and most other eukaryotes.The former is composed of four subunits, biotin carboxyl carrier protein, biotin carboxylase, carboxyltransferaseαandβ. The later is composed of a single strand peptide including four function domains, BC, BCCP, CT-αand CT-β.In plant, there are two kinds of ACCases. Gramineae ACCases both in plastid and in cytosol are multi-domain eukaryotic ACCases. It has been found that Gramineae ACCase in plastid is the target of two classes of herbicides, aryloxyphenoxypropionates (APPs) and cyclohexanediones (CHDs). These herbicides selectively inhibit the CT activity of ACCases and block the growth of gramineae grasses. Several grass species are tolerant of APP and CHD herbicides based on the presence of an insensitive form of ACCase because these herbicides are widely used. The molecular recognition mechanism between CT domain of ACCase and inhibitors is not known. In order to systemically investigate these problems, it is necessary to get purified CT domain protein of ACCase.Recently Tong L. and his companions have successfully overexpressed the homomeric ACCase CT domain of yeast in E.coli expression system. However the expression of ACCase CT domain from plant plastids has not been reported. But the homology and phylogenetic tree show that ACCase in yeast is more similar to that of wheat plastid other than to that of wheat cytosol. So we confirmed it is feasible to express CT domain of wheat plastid in vitro.Exogenous gene's expression in E.Coli including two influencing factor that is copies and translates. There are three types according to the way of expression: First, expresses in the cytosol; Second, fusion expression with other mycelium protein; Third, adds the signal peptide to the N terminatio, which can carry on the secretion expression. And the first way of expression is high efficiency, therefore this experiment uses this kind of expression. But the there are defects of the expression way that is the expressing product to be very easy to gather and forms inclusion body. Many kinds of factors influence the formation of inclusion body, such as protein's nature, yield condition (host fungus, raise temperature, pH of the culture medium and so on), expression efficiency and molecular chaperones and so on.The front part of this research is expression of truncating ACCase CT gene from Chinese spring wheat plastid. Firstly constructed recombinant plasmid T-CT2.1kb and T-CT1.9kb of Chinese spring wheat plastid ACCase CT gene truncating sequence. Our group has already constructed the pET28a+-CT2.3kb recombinant plasmid which includes Chinese spring wheat plastid ACCase CT's total length sequence. With this recombinant plasmid as template, we obtains massive gene of Chinese spring wheat plastid ACCase CT gene truncating sequence by Polymerase Chain Reaction, and clones this gene to the PMD18-T vector, in order to enhance the accuracy of clone .Constructs expression recombinant plasmid pET28a+-CT1.9kb and pET28a+-CT2.1kb of Chinese spring wheat plastid ACCase CT gene's truncating sequence. afterward expresses these two kind of proteins massively in E.coli. And simultaneously to obtain the high expression quantity and is also the soluble goal protein, has carried on some optimizations to the expression condition. The best expression condition is in 16℃the induction, derivative IPTG is divided two flowing to join or joins the sorbitol in the culture medium, expresses the fungus to use BLP, and the expression vector is pET32a+.Which the system uses is one kind of shuttle material particle, the goal gene completes connection to the vector in E.coli, then transforms into the yeast expression strain GS115 and to carry on the reorganization with its genome team. The conformity way has two kinds: one kind is the carrier linearization, forms the dissociation Aox15 the `end and 3 `ends, has the homologous double exchange with the host chromosome, in the substitution host chromosome Aox1 code area. Because the host chromosome's Aox1 structural gene is deleted, cannot code the Aox1 enzyme, therefore obtains the Muts phenotype converter. Another kind is the carrier linearization, but does not produce the dissociation 5 `end and 3 `ends, has the list exchange conformity with the host chromosome.The present paper's second part is secretion expression of Saccharomyces cerevisia yeast ACCase the CT in the Pichia yeast GS115 strain. Construction recombinant plasmid pPIC9K-CT of Saccharomyces cerevisia yeast ACCase CT to expression this protein, afterward this recombinant plasmid is transformed into yeast expression strain GS115. Using has the double exchange reorganization sub-phenotype is His+Muts characteristic screening masculine gender clone, that is these clones may not grow the mellow oxydases gene (Aox1) flaw including in the histidine culture medium to cause the yeast to be slow-growing. The masculine clone carries on the induction to express, has carried on the optimization to the culture medium pH value and the induction expression time. The best pH value is 8.0, inducing expression time is 3 days.