STANDARD PROTOCOLS FOR LYSIS OF ESCHERICHIA COLI CELLS AND PARTITIONING OF CELLULAR COMPONENTS

Lysis of E. coli Cells:

1. Harvesting the Cells: After the small-scale culture of E. coli reaches the desired optical density, harvest the cells by centrifugation.
2. Resuspending the Pellet: Resuspend the cell pellet in an appropriate lysis buffer containing protease inhibitors and lysozyme to weaken the cell wall.
3. Cell Disruption: Perform cell disruption using methods such as sonication, French press, or freeze-thaw cycles to break open the cells and release the intracellular contents.

Partitioning of Cellular Components:

1. Centrifugation: Centrifuge the lysed cell suspension to separate the soluble fraction (containing the target protein) from insoluble components such as cell debris and membranes.
2. Supernatant Collection: Collect the supernatant containing the soluble proteins and transfer it to a fresh tube for further purification steps.

Affinity Protein Purification using Strep-tag-II:

1. Equilibration of Affinity Resin: Prepare a column with Strep-Tactin resin and equilibrate it with an appropriate buffer.
2. Loading of Soluble Fraction: Apply the collected supernatant onto the Strep-Tactin column, allowing the Strep-tag-II tagged recombinant protein to bind to the resin.
3. Washing Steps: Wash the column to remove nonspecifically bound proteins and contaminants while retaining the specifically bound Strep-tag-II tagged protein.
4. Elution of Target Protein: Elute the purified Strep-tag-II tagged recombinant protein from the column using desthiobiotin or another suitable elution buffer.

Final Sample Collection:

1. Desalting and Buffer Exchange: Concentrate and desalt the eluted protein sample using methods such as dialysis or ultrafiltration.
2. Purity Assessment: Analyze the purity of the obtained sample using techniques like SDS-PAGE or western blotting.
3. Storage: Store the pure sample at appropriate conditions for downstream applications.

By following these standard protocols for lysis, partition, and affinity protein purification from small-scale cultures of E. coli, pure samples of Strep-tag-II tagged recombinant protein can be obtained.