The Protein Man's Blog | A Discussion of Protein Research

How do you break open bacterial cells in the lab? While the enzyme lysozyme is mainly responsible for lysing bacterial cells in nature, you can achieve the same effect by using particular enzymes, detergents, and chaotropic agents, and/or certain mechanical methods (e.g., sonication, repeated freezing and thawing, filtration, etc.).

Cell lysis is an essential step in the extraction and purification of intracellular proteins, and the method that you use to disrupt your cell samples can determine the success and reproducibility of your downstream applications to a great extent. Keep in mind that one vital component of the starting biological material is inevitably lost in every step of the experiment, so it’s important to get everything you need right from the start. This underscores the need of choosing the cell disruption method that will allow you to extract the best possible yield and purity of your biological materials.

Maintaining protein function and stability during the entire process of protein expression and purification is crucial. Though a lot of strategies are being implemented by many researchers, each protein has its own requirements for its stability. Some proteins are prone to aggregation, a small trigger is enough to destabilise the protein at any stage of purification. Each and every step of protein purification is challenging for maintaining stability of aggregate-prone proteins. Few strategies to be considered while purifying these sensitive proteins starting from cloning to storage of pure protein.

The main challenge during protein expression and purification is prevention of protein degradation. Whether it is an E.coli expression system or insect or mammalian, protein degradation should be minimized or avoided during purification of proteins. Protein expression at lower temperatures sometimes help in preventing the proteins from degrading. During cell lysis, proteins are exposed to harsh external conditions which result in degradation. Main reason for the protein degradation is the presence of various proteases in the cell itself.