20.109(S08):Characterize protein expression (Day5)

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.

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.

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 has a structure similar to that of histidine) can be used to elute the His-tagged inverse pericam. 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.

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.) 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. 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 (or maybe not…)

Protocols

You may find it helpful to prepare the solutions you will need for Part 3 (or at least the relevant calculations) before beginning Part 1.

Part 1: Lysis of cells producing wild-type and mutant IPC

1. You will be given an aliquot of room temperature BPER (bacterial protein extraction reagent), which also contains 0.1% bovine serum albumin (BSA, a stabilizer), and a protease inhibitor cocktail to guard against protein degradation. When you are ready to begin, add 1:1000 of cold enzyme mixture (obtained from teaching staff).
2. Per cell pellet (6 total), add 300 μL of enzyme-containing BPER and resuspend by pipetting until the solution is relatively homogeneous.
3. Vortex for 30-60 seconds.
4. Incubate the solutions (at room temperature) for 3 min.
5. Finally, spin for 3 min. at maximum speed and transfer supernatants to fresh tubes.

Part 2: SDS-PAGE of protein extracts

1. 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 emailed you 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.
2. 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 OD₆₀₀ of your WT -IPTG sample was 0.05, and the OD₆₀₀ 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.
3. 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.

Sample/Lane #	Sample Name	OD600	Volume Sample	Volume Water
1
2
3
4
5
6

1. Now add 15 μL of 2X sample buffer to 15 μL of each of your diluted lysates. Also retrieve a 15 μL sample of MW markers from the teaching faculty.
2. Boil all seven eppendorfs for 5 minutes in the water bath.
3. You will be shown by the teaching faculty how to load your samples into the gel. You might load your samples according to the table above.
4. Note the starting and stopping time of electrophoresis, which will be initiated by the teaching faculty at 200 V, and run for 30-45 minutes.
5. Pry apart the plates using a spatula, and transfer your gel to a staining box. Add Coomassie Brilliant Blue, and incubate for 1 hour.
6. The teaching faculty will show you how to transfer your gel to destaining buffer in the fume hood.
7. Replace the staining buffer after about 1 hour.
8. Tomorrow, the teaching staff will transfer each gel to water, then photograph them and post the results to the wiki. You will have a chance to physically observe your gels next time.

Part 3: Protein purification

You will process three samples (the three +IPTG extracts) according to the following procedure. Either time your spins with another group, or use a water-filled tube as a fourth, balancing tube.

All spins should be performed at 7000 rcf for 1 min.

Part 4: Protein Concentration

1. Prepare 10 mL Bradford reagent from the 5x concentrated stock by adding water.
2. Obtain BSA standards from the teaching faculty. The standards were prepared in elution buffer, since imidazole has some absorbance at 590 nm.
3. Aliquot 10 μL of each standard (0.1-1 mg/mL) into labeled eppendorfs, as well as 10 μL of elution buffer alone as a control.
4. Add 1 mL of Bradford reagent to each standard, as well as to your ?four? unknown protein samples. Incubate 10-20 min at room temperature.
5. Measure the absorbance of each sample at 590 nm.