Expression, Purification, Characterization Of PTH(1-34) And HSA-PTH(1-34)

Parathyroid hormone (PTH) is a major systemic regulator of the concentrations of calcium, phosphate and active vitamin D metabolites in vivo. Native PTH is an 84 amino acid hormone secreted by parathyroid gland, its molecular weight is about 9500 Dalton. The intact, secreted form of the hormone is generated by cleavage of a prohormone (proPTH) consisting a 31-amino-acid N-terminal extension.Native PTH regulates calcium and phosphate metabolism in the bones and kidneys. The bones are the major reservoir of calcium available to keep serum calcium levels in the normal range. Decreases in serum calcium levels stimulate PTH production, which stimulates bone turnover and release of bone-bound calcium, which in turn increases serum calcium levels. PTH also promotes renal retention of calcium and excretion of phosphate and 1-alpha hydroxylation of 25-hydroxyvitamin D, which increases gastrointestinal absorption of calcium. In 1970's, Tregear had demonstrated that the N-terminal fragment amino acids 1-34 of hPTH is fully activity in vivo.Recent 10 years, PTH was investigated intensively due to its great potential for therapeutic applications. As a PTH derivative, PTH( 1 -34 )has proved effective in increasing bone formation, augmenting bone mass, and reducing fracture rates. In 2002, a genetically engineered fragment of PTH (1-34), has been approved by FDA as the first of a new class of drugs to treat osteoporosis. The anabolic action of PTH(1-34) increases bone turnover, stimulating osteoblasts to a greater extent than osteoclasts, and reducing both vertebral and nonvertebral fractures. Whereas current PTH (1-34) injection is so expensive and patients can hardly tolerate self subcutaneous injection everyday, which limit PTH (1-34) long time clinical treatment. Therefore, it's emergent to develop some kind of economic and convenient PTH agents for optimizing treatment effect.Aimming at these problems previous content involved, we designed two schemes in following study. The first is constructing a strain of Escherichia coli, modified by recombinant DNA technology, is used to manufacture PTH (1-34) and solve the abounding stock resource in future oral and pulmonary delivery absorption of PTH (1 -34) .The second is a long-acting form of PTH(l-34) ,which has the potential to reduce discomfort by requiring fewer injections and possibly by minimizing the adverse events associated with peaks and toughs in plasma concentration that occur with daily injection. Human serum albumin (HSA) is the major protein component of human plasma and consists of a single nonglycosylated polypeptide chain of 585 amino acids with a molecular weight of 66.5 kDa . Due to its remarkably prolonging half-life, together with its wide in vivo distribution and its lack of enzymatic or immunological functions, HSA represents an optimal natural carrier for therapeutic peptides aimed at interacting with cellular or molecular components in vivo.The development of genetic engineering has opened up the possibility to obtain recombinant proteins without the danger of contamination by human pathogens, and at lower cost. HSA-PTH(l-34) fusion proteinwas produced as a recombinant protein composed of recombinant human serum albumin genetically fused at its C-terminus to the N-terminus of PTH(l-34) in Pichia. pastoris. It's will be a prospective molecular with greatly prolonged 11/2 in vivo, retaining activity of receptor stimulation and bone remodeling function. 1. Construction and characterization of recombinant Escherichia coli strain expressing PTH(l-34)1.1 Construction of recombinant pGEX-4T- PTH(l-34) expression plasmidAn artifical DNA sequence encoding PTH(l-34) was designed as E. coli. preferable codons. BamHl and enterokinase sites were introduced at the 5' terminus whereas a stop codon and EcoRI was added to 3' terminus. Following the double-stranded DNA was synthesized, it was inserted to pUC19. Then the recombinant plasmids were amplified, the target gene segment by EcoKl and BamHl digestion was inserted into linear pGEX-4T-l vector and ligated by T4 DNA ligase. The joint product was transformed to competent E.coli BL21(DE3) cells. The recombinant plasmids pGEX-4T-PTH(l-34) was screened by restriction enzyme digestion and DNA sequencing to confirm the validity of desired gene sequence.1.2 Induced expression of recombinant strain and solubility analysisE. coli BL21(DE3) transformed with pGEX-4T-PTH(l-34) was grown at 37°C and induced by 0.5mM IPTG. The collected bacteria were sonicated in the presence of 0.05% Tritox-100. After centrifugation, the supernatant and sediment sample were checked by SDS-PAGE to analysis attribution of soluble fusion protein. GST-PTH (1-34) with molecular weight 30,000 was highly expressed in E. coli BL21 (DE3) with 0.5mM IPTG inducing, which occupied 40 % of germ protein in soluble form.1.3 Purification of fusion proteinThe GST-tag in the fusion protein allowed it to be purified by Glutathione Sepharose 4B. The purified fusion protein showed a single band on 12% SDS-PAGE with the yield of 0.5 gram per liter culture was obtained after quantified by BCA protein determination method.1.4 Western blotting analysis of fusion proteinWestern boltting result displayed that GST-PTH(l-34) fusion protein can be recognized by goat anti-PTH(l-34) polyclonal antibodies .It approved PTH(l-34) gene had been inserted into expression vector correctly.1.5 Cleavage of GST-PTH(l-34) with enterokinasePTH(l-34) was cleavaged from purified GST-PTH(l-34) with enterokinase incubation at 20°C overnight. The expected peptides were collected through high speed centrifugation using MW 10000 cut-off ultrafilter tube easily. The output of PTH(l-34) is about 30mg per liter culture and purity is bove 90%.1.6 Characterization of PTH(l-34) molecular weight and primary structureRecombinant human PTH(l-34) was identified by new technique nanoESI -quadrupole-TOF tandem mass spectrometry. The measured molecular weight of PTH(l-34) was 4115.21 and measurement percent error was only 0.06 %. Digested PTH(l-34) by trypsin at 37°C over night. The tryptic digested peptides were measured and then four charged peptides were selected to measure their amino acid sequence by ESI-MS/MS. Database search results approved the primary structure of PTH(l-34) is totally correct and there is no amino acid deletion, mutation and modification in its expression and purification.1.7 PTH(l-34) activity assayThe bioactivity of the purfied fusion protein was elvaluated with classical adenylate cyclase assay. For this, we prepared fresh renal cortical membrane. The adenylate cyclase activity was assessed by measuring the amount of increased cAMP with Cyclic AMP EIA Kit.The purified PTH (1-34) stimulates adenylate cyclase in rabbit renal cortical cell membranes to nearly the same extent as synthetic PTH (1 -34) standards. The adenylate cyclase apparent activity of expressed PTH(l-34) was 2104 pmol cAMP/mg protein-30min, and that of synthetic PTH(l-34) was 2334 pmol cAMP/mg protein-30min. It indicates that the recombinant product possesses good biological activity for further research. 2. Construction and characterization of recombinant Pichia.pastoris expressing PTH(l-34)2.1 Construction of recombinant pPIC9-HSA-PTH(l-34) expression plasmidThe cDNA of HSA were transcripted from mRNA in liver tissue by RT-PCR. The PCR product of was isolated and ligated with pGEM-T vector by T4 DNA ligase. By DNA sequencing, the target gene is exactly identical with NM 000477 sequence in GenBank of NCBI. PTH(l-34) gene containing linker sequence were obtained from synthesized PTH(l-34) gene primering with special primers. The amplified recombinant were harvested, purified and cleaved with endonucleases, and the DNA segments were subcloned into expression vector pPIC9. The agarose electrophoresis of recombinant plasmid digested by Pst\ approved the target gene have been successfully inserted into expression vector.2.2 Transformation of Pichia and screening for Mut+and Muts transformantsLithium chloride transformation method was employed in producing GS115 competent cells, the linearized plasmids with Stul were transformed GS115. 30 transformants generated on RDB plate without histidine after 48h at 30°C . Picked one transformant in a regular pattern on both MM and MD plate. After growed 2-4 days at 30°C, the phenotype of transformants were screened. Mut+ transformants accounted for 90% in total transformants.2.3 PCR analysis of Pichia integrantsUsing genomic DNA isolated from Pichia clones as templates. Amplified the gene of interert by primering with a factor-up and AOXl-down. The PCR results showed that the expected size of our interest gene was about 1.9kb. This provided information that the gene of HSA- PTH(l-34) had been integrated into Pichia genome.2.4 Expression of recombinant Pichia strainSelecting several verified recombinant clones for growing at 30°Cin a shaking incubator (250r/min) . To induce expression , add 100% methanol to final concentration of 0.5%every 24h until 120h. SDS-PAGE result indicates Pichia transformed with HSA-PTH(l-34) construct secret a protein of 70kDa molecular weight and is similar to theoretic value. A time course study of expression found the yield of secret protein increased with induced time extended.The concentration of HSA-PTH(l-34) in media induced for 96h was about 400mg/L using immune precipitation method.2.5 Western boltting analysis of HSA-PTH(l-34)Western boltting result displayed that HSA-PTH(l-34) fusion protein can be recognized by goat anti PTH(l-34) polyclonal antibodies and anti-HSA serum . This approved the existence of two domains of HSA and PTH(l-34).2.6 Purification of HSA-PTH(l-34)The media induced for 96h was purfied crudely through decoloring, ammnonium sulfate precipitation and desalting with AKTA Primer. Densitometric measurement analysis of fusion protein showed its purity reached 90%.2.7 PMF characterization of HSA-PTH(l-34)Following the fusion protein digested by trypsin, MALDI-TOF-MS mass spectrograph was employed to PMF detection. Mascot database search results also provided attestation that HSA domain was exist in fusion protein.2.8 HSA-PTH(l-34) activity assayThe bioactivity assay in rabbit cortical membranes indicated HSA-PTH(l-34) expressed from Pichia possess good activation to stimulate adenylate cyclase ,its activity was 1403 pmol cAMP/mg protein-30min, but lower than that of the synthetic PTH(l-34).Altogether, concluding as following:1. A fusion protein GST-PTH(l-34) was successfully expressed and purifiede from E. coli. and proved to possess remarkable bioactivity. It provides a convenient method to produce large amount of PTH(l-34) for osteoporosis research and clinical trails. This method is simpler and more effective than chemical synthesize. In addition, the molecular weight of fusion protein was more larger than PTH(l-34),which lead to easy detection. The GST fusion tag not only enhances the solubility of recombinant proteins but also provides a good method of moderate purification. Since the fusion protein was not expressed in inclusion bodies form, a series of tough manipulation for denature and refolding was avoided. GST is an E. coli natural protein , which not only improve the translation efficiency of mRNA but also avoiddigestion of protease in the host.To identify PTH(l-34) molecular weight and whole sequence information ,we adopt nESI-MS and MS/MS mass spectrum to detecting process. These methods were proved to be fairly precise, fast, accurate and also they provide a good technical base for obtain all-rounded quality control info in the process of peptide expression and purification.2. Identified by western blotting and classical adenylate cyclase assay, genetically engeered HSA-PTH(l-34) conjugate can be efficiently secreted in P. pastoris and it retain receptor activation of PTH(l-34) in vitra.We speculated that its elimination half-life in vivo will be comparable to that of HSA and 100 fold high than that of PTH(l-34). It would have great potential in research of long-acting preparation on osteoprosis treatment.The merits of the Pichia. pastoris system lie on following points: linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors . A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of foreign gene of interest, thus enabling production of large amounts of the target protein with relative technical ease and at a lower cost than most other eukaryotic systems. As a -MF signal sequences leading , heterologous proteins were modified on the processing of the protein translation and secreted into medium, which simplify the purification procedure greatly.