Expression, Purification And Activity Analysis Of A Fusion Protein PTH-HSA In Pichia Pastoris

Parathyroid hormone is one of the most important hormones that could regulate bone metabolism. Intermittent PTH treatment at low dosage will help bone formation and increase bone mass. To prolong the stability and half-life of PTH, a fusion protein of Parathyroid hormone and human serum albumin (PTH-HSA) was expressed and secreted into the fermentation broth with a constructed recombinant Pichia pastoris.Growth curve of this Pichia pastoris was reaserched in this thesis,and the fermentation conditions of PTH-HSA expressed in the Pichia pastoris KM71 are optimized by orthogonal experiment.Result shows the optimized condition was 2.5% methonal, 30℃,pH6.5, and the suitable fermentation time was 4 days. The productivity of expressed PTH-HSA could reach 180mg/L under these fermentation conditions.Two purification routes were built up and compared.In route1, fermentation broth of PTH-HSA was concentrated by ultrafiltration and was purified by two different negative ion exchange chromatography and gel filtration chromatography in turn. The yield of protein recovery of route1 was approximately 20%.However, in route2, fermentation broth of PTH-HSA was firstly concentrated by ultrafiltration as well, and then was purified by affinity chromatography. The yield of protein recovery of route2 was approximately 61%.Purity of the prepared PTH-HSA was greater than 95%, good PTH and HSA antigenicity with prepared PTH-HSA has been identified by Westtern blotting, MW of PTH-HSA is 75kD when detected by SDS-PAGE, which were identical to predicted MW.Osteoblasts were given prepared PTH-HSA and standard PTH in intermission, investigated and compared the stimulating effects on proliferation and differentiation with the methods of MTT and PNPP.Compared the in vitro bioactivity of PTH-HSA with PTH(1-34) through detecting the expression of RANKL and OPG in bone matrix cell/ osteoblast and MSC,co-culturing UAMS32 bone matrix cell/ osteoblast and C57/Black MSC.Compared the in vivo bioactivity of PTH-HSA with PTH(1-34) by analyzing the expression of the symbolic molecule IGF-1(Insulin-like growth factor I) in bone formation.Result shows that PTH-HSA has a similar bioactivity like the natural PTH(1-34).