The Expression And Purification Of Human PTH With Site-specific Binding Tags And Biopanning Of Nanobodies Anti-PTH

Parathyroid hormone (PTH) can cause secondary hyperparathyroidism (SHPT), renal failure, even uremia when exceeds a certain amount. Many researchers have been exploring efficient methods to reduce redundant PTH in SHPT patients, including drug therapy, parathyroidectomy, and blood purification. Because drugs and surgery induce extra burden on the kidney and have other side effects, blood purification is considered as a promising way. However, the existing blood purification therapy is expensive, and shows limited ability to remove PTH because of the lack of effective adsorbing materials. Nanobodies become the best candidate of the ligants of adsorbing materials, because of their excellent characteristics. It is technically mature to select antibodies using phage display library. But the method of binding antigens is non-covalent adsorption-hydrophobic interaction in traditional biopanning, which is non-specific and gives rise to the risk of affecting key residues and morphology of ligands, especially for the small size hydrophilic proteins. Based on the situation above, we presented using site-specific binding in biopanning. We tried two methods of site-specific binding:aldehyde tag and sulfydryl tag in this paper. By comparison, we used the second method to bind antigens to secelet nanonodies. The research contains what listed below:(1) The preparation of PTH with aldehyde. First, many recombinant plasmids containing formylglycine generating enzyme (FGE) and PTH with sequence recognized and catalyzed by FGE respectively were constructed. Then two strategies of getting aldehyde were tried:in vitro catalyzation and in vivo catalyzation when enzyme and ald13-PTH were co-expressed. At last, PTH with aldehyde was produced using incompatiple plasmids co-expression systerm. But the amount of interest protein was fairly small.(2) The preparation of PTH with sulfydryl (PTH-C). A cysteine was added to the PTH by PCR and recombinant plasmid pET23a-PTH-C was constructed. PTH-C was observed soluble expressed in E.coli BL21(DE3) and accounted for25%-30%of total protein. High purity (about95%) of PTH with free sulfydryl detected by Ellman reagent was obtained after a few steps purification.(3) Nanobody selection using site-specific binding method. According to the results described above, we used antigens with sulfydryl tag in biopanning. So sulfhydryl-bind (Maleimide) modified surface plate which covalently coupled to sulfhydryl groups was used to bind antigens. First, we usd phage display together with two different selection methods: traditional non-specific binding antigen and site-specific binding antigen method,3and13fragments were isolated respectively from a non-immune library. And all of3sequences were included in the13sequences, which meant it was more efficient for the enrichment of specific nanobodies when antigens were bound by site-specific method. At the same time, the pH when immobilizing antigens site-directly was optimized:pH8.0and7.0were tested.4and13fragments were isolated respectively, but3of the4sequences were included in the13sequences. So at last we got14different sequences of nanobodies, and11of them showed high binding activity detected by phage ELISA.