Chijioke

Chijioke Okori e The Effects of Protein Expression, Purification, and Characterization 4/18/11

__Introduction__: The main objective for this lab is to view the effects of expressing, purifying, and characterizing protein. The recombinant protein would be overexpressed and there would also be a transformation of bacterial cells with a growing of a starter culture of bacteria through the expressing. Afterwards, the overexpressed protein would be purified and broken open, removing the insoluble debris by centrifugation through purification. After the purification, the samples taken from the elutions of the protein would be analyzed by gel electrophoresis through characterization.

Through the expression and purification of thousands of proteins, researchers are able to select the best methods for which to use to produce recombinant proteins. The researchers take their amount of choices needed for this process and make ‘what to try first’ strategies. Over the last ten years, researchers have targeted and purified tens of thousands of multiple proteins, including Eubacteria, Archaea, Eukarya, and many other fungal and bacterial proteins. Databases such as the Protein Data Bank, or PDB, to locate and identify the characteristics of each protein and distinguish between upon many.

Through the European Molecular Biology Laboratory (EMBL), their main tasks in expressing and purifying proteins are to: 1) providing the materials and information needed for these processes, 2) Running and maintaining equipment for cultivation of bacteria, yeast and insect cells, and for protein purification and characterization, 3) Producing proteins for the different research groups, and 4) Keeping track of the latest development in protein expression and purification and to try out new products and systems. These factors have helped these researchers in being able to find the characteristics needed for each protein and being able to identify each of them as well. This enables these researchers to be able to find certain inhibitors and drugs that can aid in a person’s health, such as through penicillin.

__Materials__:

Protein Expression: · Ice bucket · 42 degree Celsius water bath · Gas burner · 2 x 14 mL clear, sterile round-bottom tubes · 37 degrees Celsius incubator · Colirollers · 2 LB Agar plate w/o antibiotic · Competent cells on ice · Plasmid DNA on ice · LB media · SOC media · Pipette · Pipette tips

Protein Purification: · Ice bucket · Beaker of room temp. water · 1 M Imidazole · 10x PBS · 1.7 mL centrifugal tubes · 2 (two) 10mL round-bottom tubes · 4 (four) 15mL conical tubes · Bio-Rad Econo chromatography column w/ yellow cup and clear roundtop · Ring stand · Clamps · Ni-NTA resin · Benzonase (keep iced)

Protein Characterization: · Mini-PROTEAN electrophoresis tank and lid · Power supply and leads · TGS running buffer · Bio-Rad precast polyacrylamide gel · 100µL 6x gel (or sample) loading gel · Protein samples #1-6 from Expression and Purification labs · Molecular Weight standards · Plastic container w/ lid · Imperial protein stain · Whatman paper · Saran wrap · label pen

__Methods__:

Bacterial Protein Expression:

Use three different Petri dishes, one as a control group, one with DNA, and one as a fun plate. Add 25 µL bacteria to each of the groups and spin down the DNA tube, and add 2 µL plasmid to DNA culture and leave for 30 minutes and incubate. Add SOC to each tube and shake for 30 minutes. Add collirollers to each plate and shake for 2 minutes, adding the SOC/bacteria to each one. After rolling, remove collirollers and incubate overnight.

The next morning, take pictures of the plates and add 10 µL to two tubes, then touch purple colony of DNA plate and put in each tube and cap it. Incubate each tube for 8 hours and put 25 mL LB and ampicillin to flask and label them, putting 625 µL to 125 mL flask, cover and leave for incubation overnight.

The next morning, remove the flasks and take a picture of each. Use centrifuge for 10 minutes for two 500 µL culture and put in two eppendorfs, labeling them. After centrifuging, pour away remaining bacteria and put them on different tubes, and weight each out. Take picture after weighing each. Use 15 mL tube and label each, adding 2.5 mL of PBS to 50 mL tubes of pellet, vortexing them and adding lysozyme to each.

Protein Purification:

Start by thawing off 50 mL tube with suspensions of cells from Expression lab and incubate solution for 20 minutes at room temperature. After the incubation, add 2 µL Benzonase to the tube and mix by inverting tube many times and incubate again for 15 minutes at room temperature, keeping the Benzonase cool. Distribute the lysate into multiple 1.7 mL tubes and centrifuge for 20 minutes at 14,000 rpm at 4° C.

After centrifuging, take 50 mL of supernatant and dispense into microcentrifuge tubes and label sample 2, date, and initials, and store in 4° C fridge. Then, use P1000 pippetor to transfer supernatant from tubes into 15 mL conical tube, leaving cell debris behind, and dispose of tubes in Biohazard container, followed by syringing filter lysate through .22 or .45 µm filter. Then prepare buffers, wash and elution and dispense them into 15 conical tubes and add dH2 O and mix.

Add .5 mL of Ni-NTA resin/buffer and incubate for 15 minutes at room temperature and rinse 20 mL Bio-Rad chromatography Econo column with dH2O, securing using ring stand and clamp. Rinse tubes with 1 mL of 1x PBS, only after transferring resin and buffer to top of column. Let material settle for 5 minutes, remove cap and collect clear flow into 10 mL round bottom tube, labeling it "Waste". Take 50 µL sample of flow and dispense into microcentrifugal tube and label "Sample 3", along with putting initials and date. Wash Ni-NTA resin w/ 5mL of mM Imidazole in 1x PBS.

Take 50 µL sample of wash and label sample 4 and label and store in fridge. Collect buffer in 15 mL tube and label "Elution 1" and collect it turns from purple to clear and repeat, adding 5mL elution buffer and collect in another 15mL tube and label "Elution 2". Take image of both elutions. Take 50 µL samples of each elution and label "Sample 5" and "Sample 6", and put in fridge. Strip Ni-NTA wash and cap end of tubes and add 1 mL 30% ethanol in dH2O. Label columns using tape and discard waste and wash in bleach and wash each round bottom tube w/ Alconox and leave to dry. Use Nanodrop to find the absorbance for each sample and use the graphs to get the readings and paste each graph in report.

Protein Characterization:

Obtain the six samples from the purification lab and 6x gel loading buffer from the TA. With sample 1, create a counterbalance with 500 µL water in a 1.7 microcentrifuge tube and centrifuge it for 5 minutes at 5000 rpm and retain the pellet. Let out the leftover liquid in the bleach bucket and resuspend pellet in 200 µL water and 40 µL buffer. Afterwards, place all of the samples in heat bath at 95° C for 5 minutes, and then centrifuge for 2 minutes at 5000 rpm.

Get the Mini-PROTEAN tank and lid and assemble, along with obtaining the precast gel and remove the packaging for it. Clear the lanes in the tank and place the gel in a slot and close it with the comb. Use a syringe and put in the samples in each lane, first starting with protein stain and continuing on. Let the tank run for 25 minutes and set voltage to 200V. After the electrophoresis is finished, turn off the tank and disconnect leads, if not still being used. Remove the gel carefully and use tape to label the Petri dish with name and date. Throw away the water in the gel and mix with protein stain, then put on the shaker for 1.5 hours until dark bands are visible. After shaking, destain with a Kimwipe and leave again overnight on the shaker for removal of excess staining.

Next day, take a picture of the gel, putting saran wrap on top of it and post it. Then, use the Biotech lab to dry out the gel, using Whatman filter paper, and lay on the dryer and leave for 1.5 hours at 75° C. After drying the gel, take a picture of it and post it.

__Results __: = = =Lab Protein Expression:=

Figure 1a: Control Plate of the organism AMP BL21 (DE3) after being left to incubate overnight



Figure 1b: "Fun" Plate of the organism AMP BL21 (DE3) after being left to incubate overnight



Figure 1c: DNA Plate of the organism AMP BL21 (DE3) after being left to incubate overnight



Figure 2: Purple Culture in Flask after being left to incubate for about 24 hours



Figure 3: Cell Pellet after being centrifuged and Removal of the liquid (spent media)

=Lab Protein Purification:=



Figure 1: Visibility of Elutions 1 & 2



Figure 2: 1st Run of Nanodrop for Elution 1



Figure 3: 2nd Run of Nanodrop for Elution 1



Figure 4: 1st Run of UV/Vis Trial for Elution 1



Figure 5: 2nd Run of UV/Vis Trial for Elution 1

=Lab Protein Characterization:=



Figure 1: Prelimanary Picture of the Gel after an overnight washing

Figure 2: Picture taken of the gel after drying out for 1.5 hours

Figure 3: PageRuler™ Plus Prestained Protein Ladder, which is used to find the MW of a purified protein

__Discussion__: In the expression lab, there was a big difference in each of the expressions, for the control and the one with no DNA. The SOC, LB broth, and the ampicillin were all able to show how each specimen should be expressed overtime, with the DNA turning purple and the control saying relatively the same color.

In the purification lab, through the use of Nanodrop, there was not a large difference in wavelength, seeing that each sample was taken from the same elution. This could be because of the sequence given from the elutions and that the F, W, and Y nucleotides had an effect on it staying relatively the same.

In the characterization lab, it is seen that the first few proteins are easy to be seen in this gel through their stains. The gel shows the stains for which proteins through the dark visible bands seen after using the electrophoresis tank. It shows that molecular weight of sample 3 is the heaviest, while proteins such as sample 5, are not even seen very well through the gel.

__Conclusions__: It can be concluded that in each of these experiments, the expressing was more established in the plate with the control (antibiotic) rather than in the one that had none. The purifying was that the UV/Vis runs show that each of the samples were relatively the same, showing no dramatic change anywhere throughout the runs. The characterization showed that the wells give each of the stained areas to come out the way that each should after the washing and drying sections.

The sources of error for each could be through human error. In the expression, more culture and ampicillin could have been added than needed, in the purification, each of the sample from the elutions could have come out with more leftover residue (purple culture) than needed, and in the characterization, the walls of the wells could have been accidentally been pierced through, allowing for more of the sample to leak than should have.

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[|Protein Expression and Purification Core Facility]. [] (accessed April 18, 2011).