Culture Optimization

Nitrilase production as well as its catalytic properties can be improved through culture optimization at the fermentation level and gene modification at the 

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Medium development is part of the process of cell culture optimization. It can be very complicated and time consuming, and will require... 

The study of cell culture supernatants and media was chosen to exemplify the utility of the optimized quaternary HPLC method. The rate at which amino acids are consumed by protein synthesis or other metabolic pathways can be quantified by performing amino acid analysis on supernatants of the protein producing cell cultures. Optimization of target protein expression can then be achieved by feeding the culture concentrated supplements rich in those amino acids that are rapidly consumed. 

Mammalian cell culture is becoming increasingly important for the production of high-volume biopharmaceutical proteins. This is driving improvements in process efficiency. This chapter provides examples of improvements in both the creation of cell lines and in cell culture optimization, focusing particularly on experience with the glutamine synthetase (GS) expression system. 

Once the OD500 reaches 0.7-0.8, induce the fusion protein production by adding 120-200 pL of 1 M IPTG so as to obtain 0.6-1 mM final concentration for 200 mL culture. Optimal induction time and temperatures are determined empirically for each protein. Typically 2-3 h of induction at temperatures between 25 and 37 °C is sufficient. 

Van Wachem et al. (1985) reported that in their investigation of the in vitro interaction of human endothehal ceUs (HEC) and polymers with different wettabilities in culture, optimal adhesion of HEC generally occurred onto moderately wettable polymers. Within a series of ceUulose type of polymers, cell adhesion increased with increasing contact angle of the polymer surfaces (van Wachem et al., 1985). Moderately wettable polymers may exhibit a serum and/or cellular protein adsorption... 

Boskey, A. L., Stiner, D., Doty, S. B., Binderman, I., and Leboy, P., 1992, Studies of mineralization in tissue culture Optimal conditions for cartilage calcification. Bone Miner. 16 11-36. 

Most of the numerous commercially available transfection kits use cationic liposomes as transfection agent. The basic unit, Figure 2.4a, of such molecules mimics the natural lipids, which are also found as part of the cell membrane. The formulation and the exact chemistry of these molecirles is usually proprietary, hence it is difficult to postulate a mechanism or to suggest improvements of either the molecirle or the procedure. The protocols are simplified for routine use and do not present much information. Typical procedures state, for example add DNA to buffer A, add enhancer and mix with B, wait 5 minutes and add to cell culture, optimize the DNA concentration and the volume of the mix added to cells. In spite of the apparent simplicity, however, it is strongly advised to carry out the short optimization indicated by the protocol in order to obtain the maximum transfection efficiency. While kits are expensive, they require little previous experience and lead quickly to results. However, in our hands at least, these kits were not superior to calcium phosphate or PEl-mediated transfections. 

Zhu X-Y, Gong J-S, Li H, Lu Z-M, Zhou Z-M, Shi J-S, Xu Z-H. Screening, identification and culture optimization of a newly isolated aromatic nitiilase-producing bacterium— Pseudomonas putida CGMCC3830 [J]. Chin Biotechnol 2014,30 (3) 412-424. 

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