Enhanced production of hGM-CSF by medium exchange in transgenic Oryza sativa L. suspension cultures

Joo, Sung-Yeon; Lee, Kyounghoon; Lee, Yong-Il; Kim, Dong-Il

doi:10.1016/j.enzmictec.2005.11.031

Cited by 6 publications

(1 citation statement)

References 10 publications

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“…Protease activity was quantified by a modification of a published procedure (Joo et al, 2006). Substrate solution containing casein sodium (Sigma Inc.) in phosphate buffer was added to the sample.…”

Section: Quantification Of Protease Activitymentioning

confidence: 99%

Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures

Huang

Plesha

Falk

et al. 2008

Biotech & Bioengineering

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Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant alpha(1)-antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 microg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system.

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Section: Quantification Of Protease Activitymentioning

confidence: 99%