[Cloning, expression, purification of spinach carboxyl-terminal processing protease of D1 protein with hydrolysis activity and preparation of polyclonal antibody].
Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8 degrees C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of 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 x min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. 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.
Li
Hui
H
Hubei Key Laboratory of Genetic Regulation & Integrative Biology, College of Life Science, Central China Normal University, Wuhan 430079, China.
Zhang
Wei
W
Sheng
Mingxia
M
Li
Weiguo
W
Liu
Yanli
Y
Liu
Sufang
S
Qi
Chao
C
D020418:Algal Proteins; D000906:Antibodies / Q000378:metabolism*; D002268:Carboxypeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D043484:Proprotein Convertases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018749:RNA, Plant / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics
D020418:Algal Proteins; D000906:Antibodies; D018076:DNA, Complementary; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D002268:Carboxypeptidases; D043484:Proprotein Convertases; C073491:photosystem II, D1 polypeptide carboxy-terminal processing protease