T.J.S

= __ Protein Production and Purification __ =

__**Introduction:**__ The strategy of producing and purifying a protein has been perfected by laboratories that have targeted and purified thousands of different proteins from the Eubacteria, Archaea, and Eukarya (including fungal, nematode, parasite, plant and human proteins). A variety of other classes of proteins, from full-length bacterial and human proteins, to protein complexes, and even some human integral membrane proteins can also be produced in E. Coli. Overall there were three steps to this lab which included expressing, purifying, and characterizing protein samples. By overexpressing the protein of interest in bacteria, proteins that are in low abundance in their native organism can easily be purified for crystallography, enzyme inhibition assays, etc. The final purity of the protein can be optimized by controlling the ration of recombinant protein to the column size; lower-affinity contaminants can be competed with a relative excess of the histidine tagged recombinant protein. Characterizing the purified protein is some detail reduces the risk of wasting resources on protein material of inadequate quality. It also provides a means to ensure that different batches of the same protein have similar properties. This lab plays a key role with determining how the expression of proteins in bacteria can help us find a drug that could possibly target a specific bacterium. Purifying and characterizing proteins allow for one with determine how the expression of proteins in bacteria can help us find drug that could possibly target a specific bacterium. Purifying and characterizing proteins allow for one to further analyze and study human proteins without having to use human tissues. This saves time and work in the long run when searching for applicable drugs.

**__ Materials: __**  ** Protein Expression: Ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear sterile round-bottom tubes, 37oC incubator, coli rollers, 2 LB Agar Amp plates, 1 spare Agar plate without antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette with pipette tips, gas burner, and Ampicillin stock (50mg/ml). **

Protein Purification: Ice bucket, beaker of RT (room temp) water, 1M Imidazole, 10 x PBS, 1.7 ml centrifuge tubes, 2 x 10 ml round bottom tubes, 4 x 15 ml conical tubes, Bio-rad Econochromatography column with yellow cap and clear round top.

 Protein Characterization: Mini-PROTEAN electrophoresis tank and lid, power supply and leads, TGS running buffer, Bio-Rad pre-cast polycrylamide gel, 100 ml 6x gel (or sample) loading buffer, protein samples #1-6 from Expression and Purification labs, molecular weight standards, plastic container with lid, imperial protein stain, Whatman paper, saran wrap, and a labeling pen.

 Protein Expression:
 * __Methods: __ **

Day 1 – Transforming the competent bacterial cells (requires overnight transformation).

There were 3 plates involved in this lab: one: one Control plate that had no DNA to confirm that there was a clean technique and no colonies growing without the plasmid, one experimental plate with DNA, and one 'Fun' plate which was coughed on since it didn't have any antibiotic mixed in the agar (microorganisms such as yeast, bacteria, and mold will be able to grow).

Day 2-- Growing, Expressing, and Harvesting Cultures

 · (Morning)-Growing a Starter Culture o A starter protein was grown in LB along with 100 mg/ml ampicillin. Single colonies were then picked from the LB/agar and transferred to an LB/media in order to grow the bacterial cultures. · (Evening)- Protein Expression in Large Culture o <span style="color: black; font-family: 'Times New Roman',serif;">25 ml of LB along with ampicillin were added to two Erlenmeyer flasks. With the help of a shaking incubator operating at 37 degrees Celsius and 200-350 rpm, the two flasks were placed to grow for 16-24 hours.

<span style="color: black; font-family: 'Times New Roman',serif; font-size: medium; line-height: 18px;">(Day 3 or 4) <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> o <span style="color: black; font-family: 'Times New Roman',serif;">After the media was turbid and purple in color (as seen in Figure 2), a sample was taken to used later on. <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> o <span style="color: black; font-family: 'Times New Roman',serif;">An Allegra X-15 centrifuge at 3 degrees Celsius was used (ran for 10 min at 5,000 rpm) to form a purple pellet (Figure 1) which was later resuspended in buffered saline.

<span style="color: black; font-family: 'Times New Roman',serif; font-size: medium; line-height: 18px;">Protein Purification: <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · //<span style="color: black; font-family: 'Times New Roman',serif;">E. coli //<span style="color: black; font-family: 'Times New Roman',serif;">cells were lysed with lysozome. The the lysate was clarified by centrifuging samples and dispensing these samples into microcentrifuge tube <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · <span style="color: black; font-family: 'Times New Roman',serif;">After the centrifuge, a 50 micro liter sample of the supernatant was taken and labeled sample 2 <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · <span style="color: black; font-family: 'Times New Roman',serif;">The soluble fraction was then isolated by transferring liquid supernatant apart from the cell debris pellets. <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · <span style="color: black; font-family: 'Times New Roman',serif;">The next step was to syrunge filter the lysate which got rid of large particulate matter. <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · <span style="color: black; font-family: 'Times New Roman',serif;">Buffers were prepared while waiting for the previous process to complete <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · <span style="color: black; font-family: 'Times New Roman',serif;">The proteins were then purified using a using a combination of batch (Ni-NTA resin/buffer mix) and column chromotography. Two more 50 micro liter samples were taken and labeled samples 3 and 4; a 50 micro liter sample of Elution 1 and 2 were also taken and labeled sample 5 and 6. <span style="font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · <span style="color: black; font-family: 'Times New Roman',serif;">The concentration of the overall purified protein at 280 nm wavelength was measured using a Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE). The maximal wavelength (300nm) was also determined by using this instrument.

<span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;">Protein Characterization:

<span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;">Day 1-- Preparing the SDS-PAGE gel samples <span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · Samples 1-6 were collected <span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · An electrophoresis was set up and used to <span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> Day 2-- <span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: medium; line-height: 18px;"> · The gel was then dried in a vacuum set to 75 degrees Celsium on gradient cycle for 1.5 hours. 1. Day 1: Ø Prepared the SDS-PAGE gel for the six samples collected during the protein expression and purification labs and washed the gel overnight on the orbital shaker to remove the background staining. 2. Day 2: <span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">Results: Figure 1: Image of purple pellet after centrifuging. Figure 2: Image of purple culture in the flask; the purple color shows that bacteria grew. == Figure 3: Image of our plate with no DNA; no colonies grew in plate

Figure 4: Image of our plate with DNA; purple marks are all around plate.

== Figure 5: Image of our fun plate, no colonies inside of plate.

= = Figure 6: Image of Elution 1 and Elution 2. Elution 1 had a light purple color, and elution 2 was almost clear with a bit of purple.

Figure 1a: 1st Run of Nanodrop for Elution 1 with absorbance at 280 nm; yield of 0.24 mg/ml.

Figure 2a: 2nd Run of Nanodrop for Elution 1 with absorbance at 280 nm; yield of 0.24 mg/ml.

Figure 3a: Nanodrop measuring absorbance at the maximal wavelength (300 nm) of Elution 1 using UV/VIS mode at the 1st trial.

Figure 4a: Nanodrop measuring absorbance at the maximal wavelength (300 nm) of Elution 1 using UV/VIS mode at the 2nd trial.

Figure 7: Stained Gel

Figure 8: Dried gel of all samples. Proteins are clearly seen in lane 6 and 7.

Figure 9: Molecular weight standard of PageRuler Prestained Protein Ladder (Fermentas, catalog #: SM0671)


 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%;">__Beer's Law Calculations:__ A = Ebc **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">Absorbance = [(0.240 + 0.240)/2] = 0.240 <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">Extinction Coefficient = 38850 g/l = 38850 mg/ml <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">C = A/Eb = [(0.240)/ (38850mg/ml)(1ml)] = 6.18 x 10-6 mg/ml

<span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">Determining the concentration of the protein in mg/ml using Beer’s Law from the absorbance measured at maximal wavelength 300 nm: <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">Absorbance = [(0.022 + 0.021)/2]*10 = 0.215 <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">Extinction Coefficient = 118300 g/l = 118300 mg/ml <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%; line-height: 18px;">A = Ebc C = A/Eb = [(0.215)/ (118300mg/ml)(1ml)] = 1.82 x 10-6 mg/ml

__ Discussion: __

<span style="font-family: 'Times New Roman',Times,serif; font-size: 14px; line-height: 19px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0px; padding-bottom: 0px; padding-left: 0px; padding-right: 0px; padding-top: 0px;">In our expression lab, the fun plate nor the plate without any DNA had any bacteria cultures on them. By observing the agar control plates with the DNA plate, it can be concluded that there was little to no contamination on the plates. Ampicillin was key in helping prevent E. coli without the DNA from proliferation. After the bacteria was grown enough overnight, it was time for us to harvest the cells (protein expression). Different steps and processes were taken in order for this to give us the results we want. After the protein purification, a Nanodrop Spectrophotometer was used to measure the concentration of the protein at a maximal wavelength which was 300nm. When we analyzed the differences between Elution 1 and 2, we made a conclusion that Elution 2 shouldn't show any bands on the gel because of its earlier purification. <span style="display: inline !important; font-family: 'Times New Roman',Times,serif; font-size: 14px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0px; padding-bottom: 0px; padding-left: 0px; padding-right: 0px; padding-top: 0px;">Ampicillin was used to determine help prevent E. Coli without the recombinant DNA from proliferation. The purification of the targeted protein appears to be successful as seen in Figure 8: where in Elution 1 appears purple suggesting that it contains the isolated gbr22 protein. Elution 2 had a clear color suggesting the absence of gbr22. Elution 1had the most of the harvested protein which suggested a successful purification. In the (SDS-PAGE), the last two lanes were from samples of Elution one and two respectively. The faded mark in the lane with Elution 2 indicated that the protein in Elution 1 was very pure and definite. The actual molecular weight of the protein is known to be 25.79 kiloDaltons.

<span style="display: inline !important; font-size: 14px; line-height: 19px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0px; padding-bottom: 0px; padding-left: 0px; padding-right: 0px; padding-top: 0px;">After analyzing the gel

<span style="font-family: 'Times New Roman',Times,serif; font-size: 14px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0px; padding-bottom: 0px; padding-left: 0px; padding-right: 0px; padding-top: 0px;"> comparing the protein ladder with that of elution 1, a good estimation fo the purified protein's purified molecular could be about 25 or 26 kiloDaltons given the fact that the protein was successfully purified.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 110%;">**__Conclusion:__** In this particular protein expression lab, we over expressed and purified a protein in E. //coli.// Recombinant proteins were expressed in a protein and the results were the uptaking of plasmid with a purple color (from bacteria). With respect to the Nanodrop Spectrophotometer it was concluded that at a higher absorbancy, there is a higher yield pertaining to how much the bacteria was purified. It was also concluded that there is a higher extinction coeffficient at the maximal wavelength than at the 280 nm point.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. <span style="font-family: 'Times New Roman',Times,serif; font-size: 110%;"> This lab is very beneficial to Virtual Drug Screening as a whole in that it helps us create drugs to target a particular bacteria. It also helps us understand how homologous proteins bind to each other and present the drug-likeliness within a drug.All of the steps that were carried out in this lab made a difference in the final product.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 110%;">**__References:__**

<span style="color: black; font-family: 'Times New Roman',Times,serif; font-size: 110%;"> 1. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. <span style="background-attachment: initial; background-clip: initial; background-color: initial; background-origin: initial; background-position-x: 100%; background-position-y: 50%; background-repeat: no-repeat;">[] (accessed Apr 14, 2011).

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