Practical 2: Downstream processing - Cell Lysis and Centrifugation Date: 13 November 2018 Introduction: After biomolecules are grown in a bioreactor in the upstream process, they must be released from the cell to be extracted from the media. Protein extraction and purification are both part of the downstream process. About 20% of proteins in yeast cells are secreted from the cell. These proteins have a signal peptide which allows them to pass by cell receptors in the cell wall and allows them to be excreted. If the protein of interest is not secreted into the media, the cells must be disrupted to release the protein in a process called cell lysis. One solution to avoiding the whole process of cell disruption/cell lysis is to genetically modify cells to that they secrete the particular protein that is desired. But there are also many difficulties associated with this. Cell lysis is achieved by cell disruption using either physical, enzymatic or chemical mechanisms which compromises the cell's structural integrity. The disruption of microbial (bacteria, yeast and fungi) cells is more difficult than that of an animal cell because they have a rigid cell wall to protect their membrane. Bacterial cells can be lysed by sonification. A sonicator is attached to a probe which emits high frequency sound waves into the sample, causing the cells to rupture. An anti-bacterial enzyme called lysozyme can also be used to break the bacteria cell wall. For lyse yeast cells mechanically, high pressure cell disruptors are used to generate enough shear force to break their thicker cell wall. Chemical lysis with concomitant agitation in the presence of glass beads can be also be used to lyse Saccharomyces cerevisiae yeast cells. Lowering the pH will also lyse the cell wall and break the cell. The samples of yeast given in the experiment had already been through some pH changes. Ammonia, which is a strong acid, was added to decrease the pH of the solution before adjusting the pH back to 7, which is yeast optimum pH. Adding salt in to the mixture containing yeast cells interferes with the available water in the debris. This is because the salt (in the experiment, ammonium sulphate) is very soluble and dissolves in the lysate, taking up much of the available water. Because the debris has less available water, falls out of the solution. The precipitated cell debris or protein will eventually sediment out of solution under gravity, however the process can be accelerated using centrifugation, whereby the high g force causes sedimentation at a higher rate. After the cell lysis process, the protein of interest must be extracted. In the practical, two mechanisms were used in protein purification: centrifugation and dialysis. Centrifugation is used to enrich cellular membranes from sub-cellular organelles. Centrifuge tubes are filled with the broken cell extract and loaded into a centrifuge which spins the samples and subjects them to very large forces. The samples separate into a pellet at the bottom of the tube and a supernatant suspended