Cloning And Expression Of Acetyl-CoA Carboxylases CT Domain From Wheat In E.coli.

Acetyl-CoA carboxylase (ACCase EC 6.4.1.2) plays critical roles in fatty acid metabolism in most organisms. It is a biotin-dependent enzyme that catalyzes the first committed step of de novo fatty acid biosynthesis. Fllowing are the two half-reactions catalyzed by ACCase:(1) ATP-dependent activation of CO2 and attachment of the CO2 to biotin on the carboxyl carrier protein by the biotin carboxylase activity;(2)Transfer the CO2 from biotin to acetyl-CoA by the carboxyltransferase activity.So ACCase catalyzes the generation of malonyl-CoA, and this malonyl-CoA product can be used in diverse biological processes. In fact, ACCase catalyzes the committed and rate-limiting step in fatty acid biosynthesis, and is an essential enzyme in many organisms.Two isoforms of ACCase have been identified in plant cell. Heteromeric ACCase, locating at plastid in most plants, is composed of four subunits, biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), carboxyltransferaseαandβ(CT-αand CT-β). Homomeric ACCase , locating at cytosol, is composed of a single strand peptide including four function domains, BC, BCCP, CT-αand CT-β. But as an exceptional case, ACCase in plastid from Gramineae is homomeric.Evidences have indicated 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. However, the molecular recognition mechanism between CT domain of ACCase and inhibitors is not known. To systemically investigate these problems, it is necessary to get purified CT domain protein of ACCase. However, due to the low yields and instability of ACCases, there is a challenge to obtain sufficient quantity of purified enzymes by extraction from grasses. But it will be a right choice to get recombinant protein by overexpressing in vitro. Recently Tong et. al. have overexpressed the homomeric ACCase CT domain from yeast in E. coli., whereas the expression of the CT domain from plant plastids has not been reported to date. We confirmed that it is feasible to express CT domain of wheat plastid in vitro, because the homology and phylogenetic tree show that, the ACCase from yeast is more similar to that of wheat plastid, other than to that of wheat cytosol.In this study, the plastid ACCase CT cDNA from Chinese Spring wheat (CSW) was synthesized through reverse transcription (RT). Then the target gene was amplified by gene specific primers into two fragments. The total CT domain was obtained through the ligation of the two fragments by Bpu1102Ⅰ. Then the total CT domain gene was cloned into pET28a vector and transformed into E. coli. And the cloned gene was identified as the CT domain gene of wheat by DNA sequencing. Hence we constructed the recombinant plasmid of CT domain gene, named RCP18-5. The sequence of RCP18-5 was analyzed by some boi-softwares and on the internet, such as http://www.ncbi.nlm.nih.gov/blast/bl2seq/ wblast2.cgi, DNASTAR 5.00, Primer Premier 5.0 and so on. The results showed that compared the sequence homology of RCP18-5 is 98.7% and 97.9% with CSW and Tam107 wheat, respectively. However, it should be noted that, the G15 of CSW CT was a redundant base, while RCP18-5 and Tam107 have no the corresponding base. Compared with RCP18-5 and Tam107 CT, the redundant base from reported CSW leads to a mismatch of the protein N terminal five residuals. From the above results, we supposed that RCP18-5 should be regarded as a supplement and revision for reported CSW ACCase CT cDNA. The sequence of RCP18-5 was delivered to Gene Bank with an accepted NO.EU124675. Then the properties of RCP18-5 protein were predicted by bio-softwares. The results showed that the recombinant protein was high hydrophobic; the calculated molecular mass (Mr) was 88 kDa, and pI was 6.5.The recombinant plasmid was transformed into E. coli. and expressed by the induction of IPTG. To increase the expression of the recombinant protein, BL21 (DE3), BL21 (DE3) pLys (BLP), and Rosetta (DE3) were selected as expression host and the expression conditions were optimized, including induced density of cells, concentration of IPTG, temperature and time and so on. The SDS-PAGE showed that RCP18-5 could be expressed in E. coli. and, fortunately, the recombinant protein was soluble in Tris-HCl buffer. Herein, the CT domain gene was over expressed in E. coli. in a soluble form.The recombinant protein was fused with a His(6) tag and could be purified by affinity chromatography conveniently, with an purity above 80%, then the pure recombinant protein was obtained by gel filtration chromatography. The results of native-PAGE showed that the recombinant protein might aggregate to form a large polymer, indicating that it may be high hydrophobic, which is corresponding to the prediction above. It is lucky for us that the expressed RCP18-5 is soluble. However the extend or cut of RCP18-5 is not preferential for expression, which maybe the reason why the successful expression of this gene has not been reported around the world until now.The CD spectrum experiments show there are intensely changes by binding herbicide. The DSC and CD spectrum experiments show the substrate and herbicide causes the similar second structures changes of protein, which proposes the two have the similar interaction mechanisms with the protein that they locate the same region of the protein and interact with the protein in the same way. It is also found that the concentration of herbicide is much lower than the one of substrate, which proposes the interaction between protein and herbicide is much stronger.In summary, CT domain of plastid ACCase from Chinese Spring wheat has been cloned and overexpressed in a soluble state. On the basis of he results of sequence we can find that the reported partial cDNA sequence of ACCase CT could be revised and supplemented perfectly by the cloned gene RCP18-5. The 88.3 kDa recombinant protein was purified in homogeneous and the high hydrophobic protein has different properties from that of the ACCase from organisms. Hence the CT domain of wheat plastid has been cloned and overexpressed in E. coli. The purified recombinant protein can be used to study the molecular recognition mechanism between CT and herbicides, or to screen new herbicides.