Inducible Protein

Most high yield E. coli protein production systems are with the target gene under an inducible promoter. The strain is first cultivated under healthy growth conditions in order to build up the cell mass. The culture is then subjected to an induction, whether it’s a physical event such as a temperature shift or a chemical inducer such as a sugar. The target protein starts to express in large amount. The cells continue to grow, but in a somewhat slower rate. Usually in a short period of time thereafter, the expression reaches the maximum level. Cells will then be harvested and processed when the protein production yield no longer increases or cells cease to grow any further.

For our selection of strategy, the over-expressed protein should be a robust one that is easy to quantify, not necessarily a high value target. We choose E. coli lacZ encoded beta-galactosidase. It’s a widely used reporter for gene transcription and recombinant protein translation, without known toxic effect on cellular functions. It’s an enzyme that can be easily and accurately assayed, which means we may reliably capture even a small percentage of increase of beta-galactosidase expression. [A small increase, such as a mere 5%, may mean little in a laboratory setting but not so in manufacturing.

As explained above, we will create conditions to decrease cell growth around the time of induction. There are two key parameters for optimization. (1) At what time, relative to the induction point, should the induced slow growth occur (before, at the same time, or even after)? Most likely, a preemptive one should be the best. Then the question is how early the slow growth should be induced. (2) To what degree, relative to the normal growth rate before and after the beta-galactosidase over-expression, should the cell growth rate decrease? We will optimize both of these parameters in each of the following maneuvers.