In the classical protocol for inducing protein expression, bacteria are grown to early or mid-log phase and protein expression is chemically induced. Post induction, cell formation is reduced or ceases altogether as the cellular energy is dedicated to the production of a target protein. For maximum protein expression, cells are induced prior to cell saturation, requiring careful monitoring of the culture's growth. Cells are then harvested before media is depleted to avoid cellular metabolism shifting towards degradation of protein. In the auto-induction protocol for protein expression, glucose is fed to obtain high cell mass while simultaneously minimizing recombinant protein expression due to catabolite repression. Once glucose is depleted, repression ceases and the operon is induced by constituents remaining in the media. The cells continue to grow via an alternate carbon source, and a targeted protein of interest is expressed. Auto-induction protocols using lactose to induce T7 RNA polymerase and unblock the T7lac promoter are well developed. Auto-induction protocols using arabinose to induce T7 RNA polymerase and lactose to unblock the T7lac promoter have also been reported.
In this study media for auto-induction of the ara operon without lac is presented. Ara was selected for auto-induction due to several potential advantages over the lac system. First, in contrast to the lac operon gene regulation by ara repression is tight. Second, high level protein expression by arabinose induction does not preclude further cell growth. Further, induction can be cycled on and off during cell growth using glucose to stimulate catabolite repression. Lastly, the ara operon exhibits all or nothing expression, where mutant transport systems can be used to tightly control protein production.
The utility of this ara auto-induction protocol is demonstrated by producing green fluorescent protein (GFP) in fed-batch fermentation. Auto-induction media in fed-batch fermentation appears to be a novel application of the auto-induction protocol. Yields of GFP were found to exceed 250% of high cell density batch cultures in less than 12 hours. The combined fed-batch and auto-induction provides a high cell density and high protein expression system that avoids the need for human monitoring and intervention.
|Advisor:||Wang, Nam Sung|
|Commitee:||Bentley, William, Hsieh, Adam, Klauda, Jeffrey, Weigand, William|
|School:||University of Maryland, College Park|
|School Location:||United States -- Maryland|
|Source:||DAI-B 73/12(E), Dissertation Abstracts International|
|Keywords:||Arabinose, Autoinduction, Fermentation, Green fluorescent protein|
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