| Dictyostelium discoideum, also called amoeboid eukaryote is a kind of protoaoa. Its size is larger than that of other microorganisms (5-10μm in diameter), no serum is required in its culture medium and its growth rate is faster than mammalian cells. Most importantly, they can perform a whole spectrum of glycoslation modifications similar to the vertebrates. It will be widely applied as an economic and efficient expression system for the production of complicated recombinant pharmaceutical proteins (mostly glycoproteins). However, main drawbacks exist due to the low cell density and protein expression level.In this dissertation, with the target of raising the cell density of D. discoideum, the optimization of culture conditions in both flask and fermentor scales was carried out, and a dynamic growth module was proposed. Immobilization of D. discoideum was also studied both in flask scale and fermentor scale, two kinds of bioreactor with PUF or cotton fiber cloth as immobilization carriers were developed. Besides, with soluble human Fas ligand as target protein, a producing process was proposed which includes the genetic modifications of the expression system, production of recombinant protein, and primarily purifications.The optimization of D. discoideum AX3-pLu8 culture conditions was investigated. The optimal conditions were determined as following: cultivation at 22℃, pH 6.5, rotate speed 150rpm, cultivation should last for 100-120 hours; Glucose was the best carbon source with an optimal initial concentration of 15 g/l. With tryptophan 0.2g/l and cysteine 0.4g/l, the cell density could be raised even higher. The highest cell density of 3.5×107ml-1 was achieved under the conditions listed above. The culture process was then scaled up in a 5-L fermentor. The optimal conditions were pH6.5, aeration rate 1vvm, and the rotate speed varied according to the cell growth. A longer log phase of 70h and higher cell density of 3.7×107ml-1 were attained hereby. The growth kinetics was studied. A four-parameter logistic equation was derived. The results indicate that the equation can successufully simulate the whole process of D. discoideum growth. The cultivation of D. discoideum immobilized by polyurethane foam or cotton cloth in shake flask scale was studied respectively. The rotating PUF-bed bioreactor and the novel fibrous bed bioreactors (rotating style and external loop style) were developed. The highest cell density in the rotating PUF-bed bioreactor was and the highest cell densities in the fibrous bed bioreactor of rotating style and external loop style were 4.2×107ml-1, 8.2×107mL-1 and 1.37×108mL-1 respectively, far beter than that in free-cell culture (1～2×107ml-1).The original expression system was genetically modified and the recombinant plasmid vector of pMB74-shFasL-H with His-tag was constructed and transformed into D. discoideum AX3. The expression level of target protein shFasL was 157.8μg l-1. By immobilized cells cultivated in external loop fibrous-bed bioreactor, the shFasL could be increased up to 173.7μg l-1. Furthermore, with repeated batch culture, the fermentation was prolonged to over 360 hours without loss of cell density or shFasL productivity. Because of the addition of His-tag in fused protein, the primarily purification procedure of target protein was simplified and the protein with a purity of 91.0% and a recovery of 92.1% was obtained.Besides, during my doctor candidate period, I had also participated in a National "863" program project "New technologies and methods of effectively producing poly glutamic acid functional materials by microorganism". The fermentation of r-PGA was optimized, and the nitrogen medium cost of r-PGA was reduced dramatically. The process was also successfully scaled up in the 100-L fermentor, theγ-PGA productivity reached 54 g l-1. The related works are included alone in the chapter 7.
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