Extracting recombinant proteins from bacteria primarily involves breaking open the bacterial cells to release the protein, followed by purification steps.
The initial step in recovering recombinant proteins expressed within bacterial cells is cell disruption, also known as lysis. This process aims to break the bacterial cell wall and cell membrane to release the protein into the surrounding solution.
Several methods can be used for cell lysis, depending on the protein's location within the cell (cytoplasm, periplasm) and its properties. A common approach involves enzymatic methods.
Enzymatic Lysis Method
One widely used technique for cell disruption is enzymatic lysis. Enzymatic methods include lysozyme hydrolysis, which cleaves the glucosidic linkages in the bacterial cell-wall polysaccharide. This effectively weakens and breaks down the rigid cell wall, making the cell more fragile.
After the cell wall is compromised by enzymes like lysozyme, the inner cytoplasmic membrane can then be disrupted easily by detergents, osmotic pressure, or mechanical methods. This final step releases the intracellular contents, including the recombinant protein, into the extraction buffer.
Here's a breakdown of the process:
- Harvesting Cells: The bacteria expressing the recombinant protein are grown in culture and then collected, typically by centrifugation.
- Resuspension: The bacterial pellet is resuspended in a buffer solution. This buffer often contains stabilizing agents, protease inhibitors (to prevent protein degradation), and potentially components for the lysis method.
- Lysis: The cells are broken open. Using the enzymatic method:
- Lysozyme is added to the buffer. It targets and breaks down the peptidoglycan layer of the bacterial cell wall.
- Following lysozyme treatment, agents like detergents (e.g., Triton X-100, SDS) or changes in osmotic pressure are applied.
- Alternatively, mechanical forces can be used to finish disrupting the now fragile inner membrane.
- Clarification: After lysis, the cellular debris (cell walls, membranes, nucleic acids) is separated from the soluble protein extract. This is typically done by centrifugation or filtration, yielding a crude protein solution.
Other Lysis Methods
While enzymatic methods are effective, other techniques are also employed:
- Mechanical Methods:
- French Press: Cells are forced at high pressure through a narrow valve, causing shear forces that break the cells.
- Sonication: High-frequency sound waves are used to disrupt cells.
- Bead Milling: Cells are mixed with small beads and agitated vigorously.
- Chemical Methods:
- Using strong detergents or chaotropic agents directly to solubilize cell components.
- Alkali treatment (raising pH) can also lead to cell lysis.
The choice of lysis method depends on factors such as the bacterial strain, the location of the expressed protein, the required purity, and the scale of extraction.
Once the recombinant protein is released into the soluble fraction after lysis and clarification, further purification steps (like chromatography) are necessary to isolate the target protein from other cellular components.
