[Cloning and expression of human pcsk9 cDNA and preparation of its polyclonal antibody].

OBJECTIVE: To construct the prokaryotic expression vector of human proprotein convertase subtilisin/kexin type 9 (pcsk9), induce it to express and prepare the polyclonal antibody of human pcsk9. METHODS: The sequence of pcsk9 cDNA was synthesized and amplied by PCR. By means of digestion and subcloning, the sequence was cloned into the expression vector PET-28b. The recombinant plasmid of pET-28b-pcsk9 was transformed into the competent cells of E.coli BL21 (DE3), induced to express, purified and identified. The New Zealand rabbits were immunized with the purified protein. The resulted polyclonal antibody was analyzed by ELISA and Western blotting. RESULTS: The prokaryotic expression vector PET-28b-pcsk9 was constructed and expressed well in E.coli BL21 (DE3). We obtained the polyclonal antibody by immunizing the rabbits with the target protein. The purified antibody was proved to have a good specificity by Western blotting and its titer reached 1:13 200. CONCLUSION: The expression vector of pcsk9 was successfully constructed and expressed. The polycolonal antibody of pcsk9 protein was obtained as well.

Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease.

Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins.

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.