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Recombinant hPTH(l-34)ab-HSA Fusion Protein Expressed in Saccharomyces Cerevisiae

Author: LiuQingXia
Tutor: FangHuiYing
School: Jiangnan University
Course: Fermentation Engineering
Keywords: Saccharomyces cerevisiae Fusion Protein hPTH(1-34)ab-HSA Steadilyexpression intact expression
CLC: TQ460.1
Type: Master's thesis
Year: 2013
Downloads: 54
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Abstract


Human Parathyroid hormone (hPTH) is an84-amino-acid polypeptide that acts as themost important regulator of calcium homeostasis in the human body through its direct actionon bone and kidney. Clinical studies have demonstrated that hPTH(1-34) is a powerful boneanabolic agent, which is able to restore bone mineral density. It is of intriguing interest inbiomedical. The hPTH(1-34)abcombined with human serum album (HSA) could extendserum half-life. However, the N-terminal domain of fusion protein PTH(1-34)ab-HSAexpressed in the Pichia pastoris (P. pastoris) was almost degraded at random, affectingquality of fusion protein. To express hPTH(1-34)ab-HSA completely and steadily, therecombinant Saccharomyces cerevisiae (S. cerevisiae) was constructed to explore preliminary.To construct plasmid of S. cerevisiae with hPTH(1-34)ab-HAS, A genomic DNA ofPTH(1-34)ab-HSA fused to α-factor signal peptide gene was obtained by overlapping PCRtechniques, and named as α-hPTH(1-34)ab-HSA. The gene α-hPTH(1-34)ab-HSA was insertedto the plasmid pYX212. Sequencs showed that it was recombinant plasmid pYX212-α-hPTH(1-34)ab-HSA. The recombinant plasmid pYX212-α-hPTH(1-34)ab-HSA wastransformed into S. cerevisiae W303-1A by LiAc conversion method, positive colonies whichcould grow on the SC medium lack of uracil were selected. The expressed production displaysantigen of HSA and PTH by Western blot.It was explored to improve the yield of fusion protein that the constitutive promoter TPIwas replaced with the inducible promoter GAL1.The results showed that the yield of hPTH(1-34)ab-HSA increased about10%with inducible promoter. However, considering the factorsof fermentation cycle, cost savings, the constitutive promoter TPI was slightly better thanpromoter GAL1.To detect integrity of fusion protein effectively, the6×His-tag was inserted to the EcoRI restriction sites between α-Factor gene and hPTH(1-34)ab-HSA, and the recombinantplasmid pYX212-α-HIS-hPTH(1-34)ab-HSA was obtained and transformed into S. cerevisiaeW303-1A. The expressed production of HIS-hPTH(1-34)ab-HSA display antigen of HSA,PTH and HIS, which showed that the N-terminal of hPTH (1-34)ab-HSA was integral. Theresults of expressed production by recombinants, compared with P. pastoris, showed that S.cerevisiae could express intact hPTH(1-34)ab-HSA.The optimum fermentation conditions in flask culture were as follows:10%inoculum,glucose concentration to30g/L, yeast extract12g/L, liquid volume30mL/250mL,temperature30oC, pH6, shaking speed200r/min, the yield of fusion protein was8.11mg/L,which was23%higher than before.

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