Studies On Synthetic Medium For High Cell Density Cultivation Of Recombinant Dictyostelium Discoideum

The social amoeba of slime mould Dictyostelium discoideum has emerged as an attractive eukaryotic system for the expression of recombinant proteins, especially for those pharmaceutical proteins whose conformation and bioactivity depend on post-translational modifications to offer the greatest degree of product fidelity.However,the application of this system is seriously affected by slow growth rates(doubling time of 8-12 h)as well as low maximal cell densities(1-2×10⁷mL^-1)in the presence of axenic(liquid)media. Therefore,scientific efforts should be expended on the techniques for cultivating D.discoideum cells on axenic medium.In this work,a recombinant D.discoideum strain AX3-pLu8 expressing soluble form of human Fas ligand was studied.Since the medium composition has great effect on the cells growth behavior,standard synthetic SIH medium was optimized for a high cell density cultivation of D.discoideum.To understand amino acid consumption during cultivation of recombinant D. discoideum AX3-pLu8 on SIH medium,a method for determination of amino acids by reversed-phase high performance liquid chromatography(RP-HPLC) with 2,4-dinitrochlorobenzene(DCNB)derivatization was developed.16 amino acids could be well separated in 55 min,and a good linear relationship between peak area and concentration of amino acid was established with the correlation coefficients in the range of 0.992- 0.999.Analysis of culture samples of D.discoideum on SIH medium indicated that lysine was completely consumed during cultivation.Methionine,tryptophan,arginine and histidine were also utilized considerably,while aspartic acid,glutamic acid, glycine,threonine,proline and valine did not show significant demand during the cell growth.This metabolic characteristic would supply reliable data to design a more reasonable synthetic medium for D.discoideum. Starting with the standard synthetic SIH medium,the influence of different medium components on the growth behavior of recombinant D.discoideum AX3-pLu8 was investigated.It was necessary to add glucose in SIH medium, and the variation of the initial glucose concentration had a great influence on the growth behavior of D.discoideum.Maximal cell densities were obtained when about 10g L^-1of glucose was added.Adding Mg(²+)significantly improved the cell growth.At MgCl₂ concentration of 1.25 mmol L^-1,maximal cell density of 5.5×10⁷ ml^-1was achieved,which was much higher than that could usually be expected for cultivations on standard SIH medium.Ca²⁺was unnecessary to cell growth,and even showed an inhibition on maximal cell density when its concentration was higher than 3.75 mmol L^-1.Lower concentrations of Fe³⁺ showed no significant effect on maximal cell density and permitted cell growth with a higher growth rate,but higher Fe³⁺concentration showed an obvious inhibition on cell growth.Growth ceased after one to two doublings in the presence of ferric chloride for concentrations in excess of 0.3 mmol L^-1.The best concentration of Fe³⁺was close to 0.1 mmol L^-1.The requirement for vitamins showed that cell growth could hardly keep in a medium lacking biotin, folic acid or riboflavin,but the higher concentration of biotin or folic acid showed a subtle negative effect on cell growth.Optimal growth could be obtained when about 10μg L^-1biotin,0.2 mg L^-1folic acid and 2.5 mg L^-1 riboflavin were added.AX3-pLu8 did not show any requirement for VB₁₂and VB₁,and the concentration of them had little influence on cell growth.Cells inoculated into SIH medium lacking lipoid acid grew with normal growth rate, but the maximal cell density dropped drastically to 1.6×10⁷ mL^-1.The best concentration of lipoid acid was 0.4 mg L^-1.