Difference between revisions of "20.109(S08):Characterize protein expression (Day5)"
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==Introduction== | ==Introduction== | ||
− | Last time you used the lactose-analogue IPTG to induce expression of inverse pericam in DE3 bacteria. Today you will isolate IPC from the bacteria, and you will begin characterizing your wild-type and mutant proteins. | + | Last time you used the lactose-analogue IPTG to induce expression of inverse pericam in BL21(DE3) bacteria. Today you will isolate IPC from the bacteria, and you will begin characterizing your wild-type and mutant proteins. |
− | We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using (pRSET) contains six Histidine residues downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these residues, or His-tagged. Histidine has several interesting properties, notably its pKa, and His-rich peptides are promiscuous binders, particularly to metals. | + | We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=615 (pRSET)] contains six Histidine residues downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these residues, or His-tagged. Histidine has several interesting properties, notably its near-neutral pKa, and His-rich peptides are promiscuous binders, particularly to metals. (For example, histidine side chains help coordinate iron molecules in hemoglobin.) |
− | Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel beads, while irrelevant proteins can be washed away. Finally, a high concentration of imidazole (which | + | [[Image:20.109_Ni-Ag-Schematic.png|thumb|550px|right|'''Affinity separation process''' Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.]] |
+ | Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while irrelevant proteins can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors. | ||
− | + | [[Image:20.109_Histidine.png|thumb|80px|right|'''Histidine''']] | |
+ | [[Image:20.109_Imidazole.png|thumb|60px|right|'''Imidazole''']] | ||
− | After purifying inverse pericam from your bacterial lysates, you will measure the protein concentration by the Bradford colorimetric assay, named after the scientist who first [http://www.ncbi.nlm.nih.gov/pubmed/942051 published it]. The dye used in this assay is the same one you will use to stain your protein gels | + | |
+ | Prior to purifying our protein, we will lyse the bacteria, and run whole bacterial extracts on a protein gel. This procedure is called SDS-PAGE, for sodium docecyl sulfate-polyacrylamide gel electrophoresis. SDS is an ionic surfactant (or detergent), which denatures the proteins and coats them with a negative charge. Since denatured proteins are linear, they will move through the gel at a speed inversely proportional to their molecular weight, just like DNA on agarose gels. (Non-denatured proteins run according to their molecular weight, shape, and charge.) As we did with DNA gels, we will run a reference ladder containing proteins of known molecular weight and amount. When running IPTG and +IPTG samples side-by-side, you should see the emergence of a protein band at the expected molecular weight for inverse pericam, which may be very faint or non-existent in the control sample, but bright and thick in the induced sample. To visualize all the proteins released by the bacteria, you will stain the gels with Coomassie Brilliant Blue (actually, a variant called BioSafe Coomassie). This is a non-specific stain for all proteins. In a technique called Western Blotting, SDS-PAGE is combined with the use of antibodies to preferentially stain a single protein. | ||
+ | |||
+ | After purifying inverse pericam from your bacterial lysates, you will measure the protein concentration by the Bradford colorimetric assay, named after the scientist who first [http://www.ncbi.nlm.nih.gov/pubmed/942051 published it]. The dye used in this assay is the same one you will use to stain your protein gels |