Cloning, Expression, Purification Of Spinach Carboxyl-terminal Processing Protease Of D1 Protein With Hydrolysis Activity And Preparation Of Antibody

The carboxy terminal processing protease of D1 protein (CtpA), which is encoded by the CtpA gene in chloroplasts, is responsible for processing of precursor D1 protein (pD1) with a short C-terminal extension. CtpA clips the pD1 peptide extension to form the mature D1 protein (mD1), which is used in the assembly of photosystem II core. So the CtpA plays an important role in photodamage-repair cycle in photosystem II, and is predicted to be an excellent target for the discovery of a general broad-spectrum herbicide.The research aim of this work is to construct the prokaryotic expression vector of spinach CtpA and express the recombinant protein by gene engineering method. We amplified the CtpA gene with standard PCR method from spinach cDNA and constructed it into pET-23a and pET-28a vectors to give two recombinant expression plasmids. Recombinant pET-23a-CtpA and pET-28a-CtpA fusion proteins with His-tag were expressed as soluble protein in Escherichia coli BL21 (DE3) after induction with 0.2 mmol/L or 0.1 mmol/L IPTG at 8℃for 72 h. We purified the rccombinant proteins with the Ni-NTA affinity chromatography, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of pET-28a-CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg·min). We used the purified CtpA protein as antigen to immune rabbit and Balb/c mice for the production of polyclonal antibody and monoclonal antibody with the titer of 1:100 000 by ELISA respectively, and gained four hybridoma cells. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.