Zheng Chao, Zeng Xianhai, Danquah Michael K, Lu Yinghua
Dept. of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China; Scientific Research Center, Zhejiang Inst. of Medical Devices, Hangzhou, 310009, People's Republic of China.
Biotechnol Prog. 2015 Mar-Apr;31(2):424-30. doi: 10.1002/btpr.2032. Epub 2014 Dec 23.
Dictyostelium discoideum is a promising eukaryotic host for the expression of heterologous proteins requiring post-translational modifications. However, the dilute nature of D. discoideum cell culture limits applications for high value proteins production. D. discoideum cells, entrapped in sodium cellulose sulfate/poly-dimethyl-diallyl-ammonium chloride (NaCS-PDMDAAC) capsules were used for biosynthesis of the heterologous protein, soluble human Fas ligand (hFasL). Semi-continuous cultivations with capsules recycling were carried out in shake flasks. Also, a scaled-up cultivation of immobilized D. discoideum for hFasL production in a customized vitreous airlift bioreactor was conducted. The results show that NaCS-PDMDAAC capsules have desirable biophysical properties including biocompatibility with the D. discoideum cells and good mechanical stability throughout the duration of cultivation. A maximum cell density of 2.02 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 2.22 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 130.40 μg L(-1) (equivalent to a hFasL concentration of 1434.40 μg L(-1) in capsules) were obtained in shake flask cultivation with capsules recycling. Also, a maximum cell density of 1.72 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 1.89 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 106.10 μg L(-1) (equivalent to a hFasL concentration of 1167.10 μg L(-1) in capsules) were obtained after ∼170 h cultivation in the airlift bioreactor (with a working volume of 200 mL in a 315 mL bioreactor). As the article presents a premier work in the application of NaCS-PDMDAAC immobilized D. discoideum cells for the production of hFasL, more work is required to further optimize the system to generate higher cell densities and hFasL titers for large-scale applications.
盘基网柄菌是一种很有前景的真核宿主,可用于表达需要翻译后修饰的异源蛋白质。然而,盘基网柄菌细胞培养的稀释特性限制了其在高价值蛋白质生产中的应用。包裹在硫酸纤维素钠/聚二甲基二烯丙基氯化铵(NaCS-PDMDAAC)胶囊中的盘基网柄菌细胞被用于异源蛋白可溶性人Fas配体(hFasL)的生物合成。在摇瓶中进行了胶囊循环的半连续培养。此外,还在定制的玻璃气升式生物反应器中进行了扩大规模的固定化盘基网柄菌培养以生产hFasL。结果表明,NaCS-PDMDAAC胶囊具有理想的生物物理性质,包括与盘基网柄菌细胞的生物相容性以及在整个培养过程中良好的机械稳定性。在胶囊循环的摇瓶培养中,获得了最大细胞密度为2.02×10⁷个细胞/mL(相当于胶囊中最大细胞密度为2.22×10⁸个细胞/mL)和hFasL浓度为130.40μg/L(相当于胶囊中hFasL浓度为1434.40μg/L)。在气升式生物反应器(在315mL生物反应器中工作体积为200mL)中培养约170h后,也获得了最大细胞密度为1.72×10⁷个细胞/mL(相当于胶囊中最大细胞密度为1.89×10⁸个细胞/mL)和hFasL浓度为106.10μg/L(相当于胶囊中hFasL浓度为1167.10μg/L)。由于本文介绍了将NaCS-PDMDAAC固定化的盘基网柄菌细胞应用于生产hFasL的开创性工作,需要更多工作来进一步优化该系统,以产生更高的细胞密度和hFasL滴度用于大规模应用。