E. coli Expression(CRM197) Ammonium Sulfate Precipitation Collection, Redissolution, Clarification

E. coli Expression(CRM197) Ammonium Sulfate Precipitation Collection, Redissolution, Clarification

Ammonium sulfate precipitation can be used to concentrate and partially purify proteins from large amounts of crude preparations. High concentrations of saline ions in the protein solution can compete with water molecules, thereby disrupting the hydration shell of the protein surface and reducing its solubility, causing it to precipitate from the solution. Different proteins have different solubilities, so different concentrations of salt solutions can be used to precipitate different proteins. The salt concentration is usually expressed in terms of saturation, and ammonium sulfate is widely used due to its high solubility, low temperature coefficient, and minimal denaturation of proteins.

Figure 1 protein precipitation

Figure 1 Protein Precipitation

The purification of the carrier protein (CRM197) expressed in Escherichia coli was performed using the ammonium sulfate precipitation method combined with centrifugation. The E. coli fermentation broth was homogenized and clarified before protein precipitation using ammonium sulfate. The precipitate was then collected using centrifugation, and after resuspension, another round of centrifugation was performed for clarification. The two-step centrifugation process is cumbersome and results in lower protein yield.

This article mainly introduces the use of Cobetter depth filtration products to achieve the process of collecting and resuspending proteins during ammonium sulfate precipitation on the same filter, which can directly achieve clarification.

Experimental Conditions:

For the homogenized centrifuged feed with an initial turbidity of 355 NTU, after precipitation with ammonium sulfate, protein collection, online resolubilization, and clarification using a deep filtration product with a model number of 0140PC (23cm², 0.04-9.0μm). The inlet flow rate was controlled at a constant flow rate of 100-150 LMH.

Figure 2 depth filtration process

Figure 2 Depth Filtration Process

Data:

Figure 3 test data

Figure 3 Test Data

Figure 4 trend graph of filtration pressure turbidity with throughput increase

Figure 4 Trend Graph of Filtration Pressure Turbidity with Throughput Increase

Conclusion:

The depth filtration membrane showed a similar good loading capacity and recovery rate (>85%) as the centrifugation process for ammonium sulfate precipitation of the carrier protein (CRM197) expressed in E. coli. Compared to the traditional centrifugation process, it shows significant advantages in terms of operational steps and process time.