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''']] | ||
− | |||
− | + | 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 Coomassie. In acidic solution, Coomassie normally has an absorbance peak at ~ 465 nm (blue light), but this peak is shifted to 595 nm (orange light) upon binding to protein. Protein binding occurs primarily via arginine, as well as other basic and aromatic residues, as described [http://www.ncbi.nlm.nih.gov/pubmed/4096375 here]. The concentration of protein present in a sample is thus proportional to the 595 nm absorbance peak, and its absolute value can be determined using a standard curve of reference protein. We do not have a sample of inverse pericam with a known quantity of protein, so today we will use BSA as a reference protein. Because the compositions of IPC and BSA with respect to arginine may vary, this assay will really only give the relative concentrations of your protein samples, and the absolute concentrations will have an associated error. | |
+ | |||
+ | ==Protocols== | ||
===Part 1: Lysis of cells producing wild-type and mutant IPC=== | ===Part 1: Lysis of cells producing wild-type and mutant IPC=== | ||
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===Part 2: SDS-PAGE of protein extracts=== | ===Part 2: SDS-PAGE of protein extracts=== | ||
− | #Last time you measured the amount of cells in each of your samples. (If you ran cultures overnight, the teaching faculty measured the +IPTG samples for you and | + | #Last time you measured the amount of cells in each of your samples. (If you ran cultures overnight, the teaching faculty measured the +IPTG samples for you and posted the results.) Look back at your measurements, and find the sample with the lowest cell concentration. Set aside 15 μL of this sample for PAGE analysis in an eppendorf. |
#For your other five samples, you should take the amount of bacterial lysate corresponding to the same number of cells as the lowest concentration sample. For example, if the OD<sub>600</sub> of your WT -IPTG sample was 0.05, and the OD<sub>600</sub> of your WT +IPTG sample was 0.30, you would take 15 μL of the -IPTG, but only 2.5 μL of the +IPTG sample. | #For your other five samples, you should take the amount of bacterial lysate corresponding to the same number of cells as the lowest concentration sample. For example, if the OD<sub>600</sub> of your WT -IPTG sample was 0.05, and the OD<sub>600</sub> of your WT +IPTG sample was 0.30, you would take 15 μL of the -IPTG, but only 2.5 μL of the +IPTG sample. | ||
#Next, add enough water so the each sample has 15 μL of liquid in it. You might use the table below to guide your work. | #Next, add enough water so the each sample has 15 μL of liquid in it. You might use the table below to guide your work. | ||
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! Sample Volume | ! Sample Volume | ||
! Water Volume | ! Water Volume | ||
+ | |- | ||
+ | | 0 | ||
+ | | pre-stained ladder | ||
+ | | --- | ||
+ | | --- | ||
+ | | --- | ||
|- | |- | ||
| 1 | | 1 | ||
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| | | | ||
| | | | ||
+ | |- | ||
+ | | 7 | ||
+ | | unstained ladder | ||
+ | | --- | ||
+ | | --- | ||
+ | | --- | ||
|} | |} | ||
</center> | </center> | ||
− | #Now add 15 | + | #Now add 15 |