ALICE+C

__Title: __

Protein Expression, Purification, and Characterization Make title little more creative/detailed

__Introduction: __

Because proteins are often difficult to express in large enough quantities in human tissues, many studies involving proteins use plasmid vectors to insert a strand of DNA into a bacterial genome to express. Non-virulent strains of //E. coli//bacteria are often used for this task because over 50% of Eubacteria and Archaea and 10% of Eukarya proteins can be expressed in these species [1]. Once the bacteria are grown, the cells are lysed and the target protein is extracted and purified through filtration steps, which vary by protein. This process is aided by various tags that are added to the end of the target protein in order to separate it from non-target proteins [1]. The identity of the resulting sample must then be verified to ensure an accurate study.  Protein expression, purification, and characterization is crucial in many fields of biological and biomedical research and allow for further studies of the proteins through crystallography, enzyme inhibition assays, etc.

In this series of experiments, the BL21 (DE3) strain of //E. coli//was selected to express a pGEM-gbr22 protein. This plasmid, or circular sequence of DNA that is capable of automatically replicating in a host cell, may be translated to a fluorescent protein that originates from a purple coral in the Austrailian Great Barrier Reef. It was selected for its color and contained both a gene for ampicillin resistance and a hexa-histidine tag at the C-terminus to facilitate purification. Lysozyme, which digests cell walls, and cyanase, which degrades nucleic acids, work to separate the target protein from other portions of the bacterial cells. They are used in this lab to facilitate the removal of insoluble cell parts during purification.  If the plasmid is successfully implanted in the //E. coli//bacteria and the resulting purification techniques are completed under sterile conditions, the characterization of the protein should show a pure sample of the target pGEM-gbr22 protein.

 __Materials & Methods:__

For expression, two samples of BL21(DE3) were iced with plasmid, heat shocked, and returned to ice. The solutions were then transferred to SOC media, shaken in an incubator, and spread on separate LB/amp agar plates with colirollers. The next morning, one bacterial colony was transferred to LB/amp media to shake in the incubator for 8 hours. The starter culture was then moved into a LB/amp flask for further growth. Later, the sample was spun to isolate the pellet, which was resuspended in PBS buffer.

For purification, lysozyme and cyanase We used benzonase. Include how much  were added to the bacteria. The solution was passed through a 0.45um Millex HA syringe filter and further purified with Ni-NTA column chromatography. A Ni-NTA resin/buffer solution was mixed with the bacteria while samples of water and 20mM imidazole were used to flush impurities. Two 250mM imidazole solutions were then used to remove the target protein from the nickel. The absorbances of the samples were measured with the Nanodrop® spectrophotometer at 280nm and maximal wavelength. Mention company and it's location for any specific equipment you use! (ex. spectrophotomer, PAGE ladder, etc.)

For characterization, the samples taken throughout expression and purification were heated and centrifuged with loading buffer, loaded into individual gel cells, and subjected to an electric current in an electrophoresis tank. The gel was then cleaned in nanopure water and stained with imperial protein stain on an orbital shaker. After destaining, the gel was dried on the drying bed and documented. Mention PAGE ladder

 __Results:__













Label the different lanes in the gel! Also include an image of the PAGE RULER prestained from ThermoScientific website. To determine the concentration of elution 1, use Beer’s law:

A = εBC

<span style="font-family: 'Times New Roman',serif;"> __For the 280 nm wavelength reading:__

<span style="font-family: 'Times New Roman',serif;">0.497 = 38850M-1cm-1 (1cm)(C)

<span style="font-family: 'Times New Roman',serif;">C = 0.3299mg/mL

<span style="font-family: 'Times New Roman',serif;">Yield = 0.3299mg/mL x 5mL

<span style="font-family: 'Times New Roman',serif;">= 1.649mg

<span style="font-family: 'Times New Roman',serif;"> __For the 574 nm wavelength reading:__

<span style="font-family: 'Times New Roman',serif;">0.086 = 118300 M-1cm-1 (0.1cm)(C)

<span style="font-family: 'Times New Roman',serif;">C = 0.1875mg/mL

<span style="font-family: 'Times New Roman',serif;">Yield = 0.1875mg/mL x 5mL

<span style="font-family: 'Times New Roman',serif;">= 0.938mg

<span style="font-family: 'Times New Roman',serif;">The calculated yields are significantly different because different amino acids absorb light at the two wavelengths. Only tyrosine, tryptophan, and cystin absorb 280 nm while the rest of the protein absorbs 574 nm. The 574 nm calculation is likely more accurate because it accounts for the whole protein rather than just the three amino acids.

Why doesn't the yield on here match what's on the front page (0.56 mg)?? <span style="font-family: 'Times New Roman',serif;"> __Discussion:__

<span style="font-family: 'Times New Roman',serif;">Potential sources of error from this lab include that cell debris could have been included when the debris was separated from the solution containing the target protein. This could influence the absorbance values calculated from the spectrophotometer and alter results. Additionally, the volume of the elution solutions was not measured but assumed instead. Though 5mL of the buffer was passed in the column, some could have stuck to the apparatus and decreased the actual volume, which means that the assumption was high.


 * **<span style="font-family: 'Times New Roman',serif;">Sample number ** || **<span style="font-family: 'Times New Roman',serif;">Contents ** ||
 * <span style="font-family: 'Times New Roman',serif;">1 || <span style="font-family: 'Times New Roman',serif;">Solution with bacteria before centrifugation ||
 * <span style="font-family: 'Times New Roman',serif;">2 || <span style="font-family: 'Times New Roman',serif;">Bacterial cells with lysate solution ||
 * <span style="font-family: 'Times New Roman',serif;">3 || <span style="font-family: 'Times New Roman',serif;">Flow-through waste from 1xPBS rinse ||
 * <span style="font-family: 'Times New Roman',serif;">4 || <span style="font-family: 'Times New Roman',serif;">Wash solution ||
 * <span style="font-family: 'Times New Roman',serif;">5 || <span style="font-family: 'Times New Roman',serif;">Elution 1 ||
 * <span style="font-family: 'Times New Roman',serif;">6 || <span style="font-family: 'Times New Roman',serif;">Elution 2 ||

<span style="font-family: 'Times New Roman',serif;">The wash buffer, 1x PBS and 20mM imidazole, and the elution buffer, 1xPBS and 250mM imidazole, differ in ability to bind to the 6x HIS tag. The lower concentration imidazole wash solution was low enough to remove only the large debris protein in the mixture. The higher concentration elution buffer had a high enough binding affinity to the tag to remove the target protein from the Ni-NTA. Good job with the error analysis! But also include other info (like discussion questions!) in this section.

<span style="font-family: 'Times New Roman',serif;">The purity of the protein was approximately 30%, which is quite low. This may have been due to the limited purification technique used, as the nickel could have attracted other proteins.

MISSING: why we use lysozyme, benzonase? How does HIS tag system work? What was in the sample after each step? size of your protein compared to size expected in protein purification? Difference between wash and elution <span style="font-family: 'Times New Roman',serif;">?

<span style="font-family: 'Times New Roman',serif;"> __Conclusions:__

<span style="font-family: 'Times New Roman',serif;">In these labs, a target protein was inserted into a host bacterium, over-expressed, purified, and characterized. The transformation, extraction, and identification techniques used in the lab will allow us to make significant amounts of a target protein, harvest it, and use it for further testing and facilitate our independent projects by separating a sample of proteins to test and be sure the target protein is being studied.

Mention enzyme and inhibition assays. Mention protein yield/other key findings.

<span style="font-family: 'Times New Roman',serif;"> __References:__

<span style="font-family: 'Times New Roman',serif;">1. Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. //Nat Methods// 2008, **//5//** **(2), 135-46.**

<span style="font-family: 'Times New Roman',serif;">2. Protein Expression and Purification Core Facility. <span style="font-family: 'Times New Roman',serif;"> [] (accessed April 15, 2013)