Experiment+12

Recombinant Protein Expression/Immunoprecipitation

From pgs 339-344

Special care must be used when preparing the plasmid of foreign DNA A recombinant protein can reach as high as ~40% of total cellular protein. Although high is not all always a good thing, as the bacteria may form __inclusion bodies__ from your protein. However, some of the inclusion bodies can be renatured by centrifugation and the addition of a denaturing agent then slowly removing the denaturing reagent. One might expect inclusion bodies to be undesirable but they can be beneficial in some circumstances. because they can save time in purification steps.
 * Gene Expression in Prokaryotic Organisms**

__inclusion bodies__ - Aggregated clumps of denatured protein. []

Engineer correctly -> avoid inclusion bodies and encourage secretion -> release from cell wall with osmotic lysis Benefit: You don't pop the cells so you don't need to purify out all the other things inside them.
 * Host Cell Secretion of Protein**

- Secrete the protein - Add protease resistant a.a. sequences to your protein. - Make a __hybrid/fusion protein__, remove the undesired bacterial protein later.
 * Reduce intracellular degradation.**

__hybrid/fusion protein__ - your protein tagged(covalently bound)with a protein of bacterial origin

String of 6 histidines on the N terminus of the protein -> binds to nickel. You can then use nickel bound to a bead to pull your protein out of solution. Other similar systems: Polyarginine - add to C-terminus, makes the protein basic -> bind to cation resin. Flag - short amino acid sequence attached to N terminus -> binds to monoclonal antibody
 * Histidine-Tagged Proteins**