Studies On Expression And Purification Of Recombinant Human Parathyroid Hormone 1-34 In Pichia Pastoris

Parathyroid hormone (PTH) is secreted by parathyroid cell, and it is a kind of straight-chain peptide with 84 amino acid. PTH is the capital hormone to retain the balance of calcium and phosphor. PTH can regulate the metabolism of Ca2+, PO43- with Calcitionin and Vitamin D by recognizing the specific receptors which are on the suface of bone cell, small intestine cell and kidney cell. By researching we found the N-terminal fragment amino acids 1-34 (PTH 1-34) can play all function of PTH. Now PTH 1-34 has been approved by FDA in USA as a new class of drugs to treat osteoporosis. Whereas it is so expensive and needed everyday, and the domestic clinical study is very few. So it is necessary to adopted gene engineering technology to genetate PTH 1-34 in order to reduce the high cost and gain generous protein production. The reported study of PTH and the characteristics of eukaryocyte expression system-Pichia Pastoris system were reviewde in this article to construct the recombinant yeast expression system with PTH 1-34 and study the expression conditions and purification.1. Shake-flask expression of PTH 1-34 and selection of high-level expression coloniesWe changed the genetic codons of PTH 1-34 to Pichia pastoris favorite codons by artificial synthesis. Cloning vector is pBluescriptⅡSK(+), cloning site is XhoⅠ/EcoRⅠ.The primer inserted XhoⅠtarget site,Kex2 in 5'end,terminal codon taa and EcoRⅠtarger site in 3'end. Kex2 is essential to cutαsignal peptide efficiently. Then we got the recombinant plasmid pBluescriptⅡSK(+)-PTH (1-34).After XhoⅠ– EcoRⅠdigestion, the PTH 1-34 gene was cloned in frame into the pPICZαC vector between the XhoⅠ(5' end) and EcoRⅠ(3' end) restriction sites to generate the plasmid pPICZαC-PTH (1-34) . After confirmation by endonuclease digestation assay and sequencing, recombinant vector was linearized by SacⅠ, then transformed into the X-33 via electroporation. Transformed yeasts were plated on YPD plates containing Zeocin to isolate-resistant clones. After clones were cultured for 72 hours at 28℃, 9 single clones were picked into culture. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the primers.2. Expression and identification of PTH 1-34Proliferate the PCR tested positively yeast clones in culture fluid BMGY, then induced the expression of PTH 1-34 with 0.5% methanol everday for 8 days. Perform SDS-PAGE, ELISA to identify PTH 1-34, then we could get the most expression level strain.3. Optimization of expressional conditionsThe expression level of PTH 1-34 was evaluated by SDS-PAGE and ELISA analysis of expression medium supernatant per day past 8 days induction by methanol at pH 3.5-7.5. Levels of PTH 1-34 increased gradually after induction and reached the peak at day 7. The expression level reached a maximum when the pH value of the medium was 5.5.4 Study on purification of PTH 1-34The most high-level expression colony was plated on YPD plates containing Zeocin and proliferate in 10ml BMGY, after 24h proliferate in 200ml BMGY, finally, proliferate in 2L BMGY. After exhastion of glycerin, inducing the expression of PTH 1-34 with 0.5% methanol in pH 5.5 for 8 days. We can see the level of expression is higher and higher by SDS-PAGE and ELISA analysis of expression medium supernatant per day past 8 days.When the level of PTH 1-34 reached the peak, we centrifuge the culture. With an AKTA Explorer 100 chromatography system, the optimal concentration of NaCl for elution was 0.3 M and the optimal pH for blinding was 4.0. The PTH 1-34 solution was purified with cation exchange chromatography (SP Sepharose XL) and reverse phase chromatography (Source 30). Following these processes, we could get a high purity PTH 1-34 in high concentrations of methanol. In order to remove the methanol, the solution must be futher purified by vacuum distillation. Then we could get a more than 80% purity protein of PTH 1-34.In conclusion, our studies succeed in secreted expression of PTH 1-34 in Pichia pastoris. Established initially expression system of PTH 1-34. We also study the way of purificate the protein production. This can provide a basis for the developing research of fermentation and purification.