LindsayG

=Bacterial protein expression, purification and characterization.=


 * //Introduction//**: There are three steps in this lab, to express, purify and characterize a protein and then analyze the protein samples that were collected during the expression and purification labs. This process inclues the preparation of a clarified samples of soluble protein from the source material for further purification and the removal of particulate matter which are not compatible with chromatography [1]. Trying to express certain recombinant proteins in bacteria has been used several times throughout biological and biomedical science. There have also been many questions concerning in which cell a certain protein should be expressed in and if in bacteria which strain should be chosen. Purifying is also important in this process and can be optimized by controlling the ration of recombinant protein to the column size. It is also beneficial to determine the amount of the soluble target protein to be loaded. Characterizing a purifies protein can reduce the risk of wasting resources on protein material of inadequate quality. This also provides means to make sure that different batches of a protein have similar properties [2]. In this lab, a protein was overexpressed in E.Coli and was purified byremove the insoluble celss debris by centrifugation and lastly characterize the protein by using gel electrophoresis and yield the final purified protein product. This lab is very important as it contributes to how to express certain proteins in bacteria that could help us know something about a drug that could target that particular bacterium.

**//Material & Methods//**: For part 1 of this lab (**protein expression**), the following are needed: ice bucket, water bath, gas burner, clear, sterile tubes, incubator, colirollers, agar amp plates, the competent cells on ice, plamid DNA and LBmedia and pipette and pipette tubes.

Transfrom the competent bacterial cells by putting appropriate amounts of each SOC mixture (one with bacteria and one without) in each plat. The next day grow the starter culture and later on in the evening make a large culture for overnight expression. On day 3, harvest the cells, take a picture of these cultures in the flask. Resuspend the cells in phsphate buffered saline by adding lysozyme to a final concentration of 1 mg/ml.

For part 2 of this (**protein purification**) the following are needed: ice bucket, Imidazole, 10x and 1x PBS, centrifuge, round bottom, conical tubes, resin and benzonase.

Lyse the E.Coli cells and then clarify the lysate and then isolate the soluble fraction. Then syringe filter the lysate and make sure to wear goggles and lab coat because the filter can burst. Prepare the buffers; there will be two buffers the wash and elution buffer. To make the elutions, transfer the resin and buffer to the top of the column by pouring and allow the resin to settle evenly. The liquid collected will be elutions 1 and 2 and should be clear. Now use the Nanodrop spectrophotometer to estimate the concentration of the final purified protein. Measure the wavelength at 280 nm and also measure at the maximal wavelength using the spectrophotometer. From this determine the yield of purified protein collected.

For the last part of the lab (**protein characterization**), the following are needed: electrophoresis tank, TGS running buffer, polyacrylamide gel, loading buffer, protein samples for previous 2 labs, standards, and imperial protein stain.

Prepare the SDS-PAGE gel samples. Then assemble the electrophoresis module. Load the sample by using a needle and syringe and take up a milliliter and then inject this into the well forcefully to clear it out. To stain the gel add the imperial protein stain to completely cover the gel. Wait for 1.5 hours until dark bands are visible. Destain the gel after this. Dry the gel the next day. Take a piece of Whatman filter paper and put the gel on top of this. Cover it with Saran Wrap and lay on drying bed. This should be done for 1.5 hours.

Part 1: Protein Expression
 * //Results//**:





Part 2: Protein Purification





Part 3: Protein Characterization





Pre-Lab Calculations 5 mL (100) = 500 mL 500/50000 = 0.01 = 10 microliters (1*10^-5 microliters)*5 =50 microliters 10*V - 10 mL (1x) V= 1 mL + 9 dH2O
 * Expression**:

Added stock for lysozyme

(50 microgram/microliters)(x)= (1 mg/mL)(2.5) 50x = 2.5 mg/mL 2500 microgram/50 microgram/mL = 50 microliters **Purification**: Pre-Lab Calculations 1. (10X PBS)(X mL) = (1X PBS)(10 mL) X = 1 mL 10X PBS 9 mL water 2. (1 M Imidazole)(X mL) = (20 mM Imidazole)(10 mL) X = 0.2 mL = 200 μL 1 M Imidazole 200 μL 1 M Imidazole + 1 mL 10X PBS + 8.8 mL water (Wash Buffer) 3. (1 M Imidazole (X mL) = (250 mM Imidazole)(10 mL) X = 2.5 mL 2.5 mL 1M Imidazole + 1 mL 10X PBS + 6.5 mL water (Elution Buffer)

Maximal Absorbption Wavelength: 574 Extinction coefficient @Max wavelength: 118300 P280 nm Ext Coefficient: 38850 Molecular Weight of gbr22: 25794.2 g/mol

To determine the concentration of aprotein using 280 nm WAVELENGTH Avg Abs, P-280 A= ebc (.1005) = 38850*1*C C=2.587*10^-6 mg/mL

To determine the concentration using MAXIMAL WAVELENGTH A=ebc .975=118300*1*C C=8.24*10^-6 To determine the yield @ 280 nm V=4.5 mL A=2.587*10^-6 mg/mL mg= 1.16*10^-6 mg

The calculated yield @ the maximal wavelength is 3.79*10^-5 Pre-Lab Calculations: 1. 5x TGS = 500 Ml (1X) TGS= 100 mL, 400 mL of H2O 2. (6x)(V2) = 50 M (1x) 8.333 microliters=(1 mL/1*10^-3)= 8.333*10^-3 mL
 * Characterization**:

//**Discussion**//: Figure 1 represents the two cultures of bacteria expressed in the first part of the lab. There were however some errors that happened in this experiment. When bacteria from each tube (DNA and control) in each of the agar plates, but when cultured overnight bacteria did not grow onto one of the plates. So instead, a culture of bacteria was taken from another group and used to complete the remainder of the lab. The purple cultures in each of the flasks mean that the bacteria has grown by taking up the plasmid. Several images were taken of the bacterial agar plates, grown culture and the cell pellets. Certain solutions were centrifuge and the supernatent was drained off which left pellets, as seen in figure 2. The masses where taken. The pellet A weighed 0.67 grams and pellet B weighed 0.94 grams. Figure 3 shows the original agar plates; as seen there should be purple cultures, which mean that the bacteria took up (or expressed) the plasmid. Figure 4 shows the elutions from the 2nd part of the lab (purification). Both elutions should be clear or transparent; one of the elutions was slightly purple. Figure 5-8 show the results from the nanodrop spectrophotometer. These analyses had to be redone for a second time because the original had a negative concentration. The graphs above show a correct reflection of how the graphs should look like (another groups elutions were used and hence calculations). There were also some errors that occurred in this lab. When clarifying the lysate and dispensing some of the lysate into the syringe filter, some of the supernatent spilled onto the ground. Also the wash was not transparent like it should have been. This may have been because the tubes were not mixed properly. Figure 9, 10 and 11 are from the protein characterization. Figure 9 shows the stained gel with respective bands. Figure 10 shows the finished dried gel labeled with what was in each well. Lane 1 held the protein ladder, and lanes 2-6 held each sample. Figure 11 shows a picture of the molecular weight standard used. The molecular weight standard used was 10-170 kDa. there were no protein bands in lane 5 because there was not any elutions to put in that lane. There was an error in sample loading and power conditions. Something happened with the machine that loads the samples. Also thre were no bubbles being seen the first time the samples were loaded and had to be run again. This whole 3-part lab would have to be redone because a lot of the protein was lost throught he whole process. Sample 5 probably was not pure. The molecular weight cannot be estimated because a lot of the protein was lost.

//**Conclusion:**// Bacteria acts in different environment manipulating different facts. Recombinant proteins were expressed in a protein and saw how the plamid uptook the DNA through the color of bacteria (purple). At a higher absorbancy, there is a higher yield than at the 280 nm point regarding how much the bacteria was purified. From this protein purification lab it was also concluded that there is a higher extinction coeffficient at the maximal wavelength than at the 280 nm point. The molecular weight of the gbr22 protein could not be identified because of the amount of protein that was lost through out the whole process. This is beneficial to the virtual drug screening stream because it helps us develop drugs to target a specific bacteria and learn how it binds through how it uptakes certain plamids from a protein.

//**References**//: 1. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed Apr 16, 2011). 2. Protein production and purification. //Nat Methods//. [Online] **2008,** //2,// 135-46 [] (accessed Apr 16, 2011).