KARINNA+L

__ Title: __

Quality not Quantity: Purple protein (pGEM-gbr22) expression, purification and characterization.
__ Introduction: __ ====In the 21st century, a significant amount of time is spent doing research with the sole purpose of maximizing production of drug discovery or specialized procedures in biotechnology. The most challenging part of doing research is to know where to begin which is why a determined sequence of steps must be followed to ease the process. The objective of this lab is to introduce procedures used to maximize productivity which is done by the expression, purification and characterization of a protein. In an attempt to speed up protein expression, a recombinant protein was created by the insertion of a plasmid DNA (pGEM-gbr-22) encoding the sequence of gbr-22 which is a purple fluorescent protein trying to be expressed into a host cell in this case E.Coli bacteria (BL21). E.Coli is used since it is ‘…fast and inexpensive to test a wide variety of possible strategies (strategies?) in //E. coli//, and one can complete a fairly comprehensive analysis within a relatively short period of time…”. [1] (I'm not sure if you should be using quotes) Once the protein is expressed, the process of purifying the protein is next which in a way is simply the cleansing of the protein. Immobilized metal affinity chromatography (IMAC) is used in this process since“…the final purity of the protein can be optimized by controlling the ratio of recombinant protein to the column size; lower-affinity contaminants can be competed with a relative excess of the histidine-tagged recombinant protein…” [2] In addition, once the protein is purified characterization will occur in order to measure purity and concentration of the protein. The hypothesis is that gbr-22 will be successfully expressed and purified in BL21 through the process of IMAC and once characterization occurs 75% purity will be the result. (Good intro, but try to state things without quotes) ====

__ Materials & Methods: __

[Protein Expression]
==== Day 1: Three plates were labeled accordingly: one experimental plate (Bl21 DE3-pGEM-gbr22), one control plate (BL21 DE3-No DNA) and one fun plate. 25uL of E. Coli bacteria was added into two transformation tubes along with 1uL of plasmid DNA added to the DNA tube only. Next, the samples were placed on ice for 30 minutes; heat shocked at 42 ° C for 45 seconds in a water bath, placed in ice for 2 minutes and 200uL of SOC media was added to the tubes. Next, samples were shaken in an incubator for 30 minutes at 37 ° C and about 250 rpm, 5-6 coli rollers (Things like the number of coli rollers are too much) were added and 50uL of the bacteria/SOC mixture were pipette to the different plates and spread. The plates were then stored in a 37 ° C incubator overnight.==== ==== Day 2: 10uL of ampicillin were added to two tubes of 5 mL LB broth along with a single colony of bacteria that expressed the gbr-22 protein was placed into a flask in the shaking incubator for 8 hours at 37 ° C at 200 rpm. 25mL of LB broth and ampicillin were then transferred to two Erlenmeyer flasks along with 0.625mL of starter culture and then placed in the shaking incubator at 37 ° C and 200 rpm for 16-24hrs.==== ====Day 3: Bacteria was poured into a 50 ml conical tube and centrifuged in the Allegra X-15 centrifuge set to 4 ° C for 10 minutes at 5000 rpm. The pellet and 50 uL of the liquid (Sample 1) were saved. 2.5mL of 1x PBS solution was added to the 50 ml tube containing the pellet. The tube was vortexed; 50ug/ul lysozyme was added to a concentration of 1mg/mL and vortexed again. Sample was placed in the -20C freezer.====

[Protein Purification]
====2.5 ml of suspension was thawed, incubated for 20 minutes, 2uL of Benzonase was added, incubated for another 15 min, distributed into several microcentrifuge tubes, centrifuged for 20 minutes at 14,000 rpm at 4C and 50uL was saved as sample 2. Sample was filtered using a 0.22 PES 5mL syringe filter, 0.5mL of Ni-NTA was added and incubated at room temperature for 20 minutes. 20mL Bio-Rad chromatography Econo column was rinsed and then ran through with a wash and elution buffer. A nanodrop spectrophotometer was used at 280 nm and 574nm to measure concentration in Elution 1.====

[Protein Characterization]
====Sample 1 was micro centrifuged for 5 min at 5000 rpm and the pellet was resuspended in 200uL of water. 6x loading buffer was added to each of the samples 1-6 and they were placed on a heat block at 95C for 5 min, centrifuged again for 2 min at 5000 rpm, electrophoresis module was set up, 7 uL of the MW standard (Page-Ruler, #26616,Thermo Scientific) was used as ladder in well 1. Samples 1-6 were added after and the gel ran for 25 minutes. Afterwards, the gel was stained with imperial protein stain, and destained overnight. The next day, the gel was dried at 75 ° C for 1.5 hours and analyzed after.====

(Very detailed! Most is good, but some is too much info)

__ Results: __ Figure 1- Experimental plate of BL21 E.Coli host bacteria expressing about 60 pGEM-gbr22 colonies growing on ampicillin treated petri dish after overnight incubation. (Where are the two other plates?)

Figure 2 -Harvested cell in overexpressed purple culture in flask of gbr22 protein after 24 hour incubation. (What was the RPM and temp?)

Figure 3- Wet pellet containing gbr22 with a weight of 0.53 grams after centrifugation of large culture of BL21(DE3).

Figure 4a- Elution 1 containing gbr22 protein Figure 4b- Elution 2 with a clear color due to no after purification. A hint of purple is present. presence of gbr22 after purification. (What's the buffer that its in?)

Figure 5 -Nanodrop spectrophotometer absorption screen shot at 280nm for Elution 1 containing gbr22 with an absorbance of 0.168.

Figure 6- gbr22 protein g el gel after electrophoresis with an approximate MW of 25 kDa. Purity for Elution 1 is about 50%. Gel image is flipped. (Please write out what each lane is in the caption) Figure 7- Molecular Weight Standards of diverse protein sizes stained in different colored dyes measured in kDa. (Company name?)

Beer's Law A=εBC, A =.168B = 1cm Extinction coefficient: 33850 M-1 cm-1 MW of gbr22: 25,794.2 g/mol C = (0.168)/(38850) = 4.324E-6 M 4.324E-6 M (.005L)(25,794.2g/mol) = 0.558 mg gbr2

__ Discussion: __

The goal for the lab was to produce a gel (figure) from the expressed and purified protein.
However, there are two bands present in Elution 1 which is were the protein is found which means that contamination is present and purity is about 50%.. Due to contamination, the lab can definitely be improved through a more careful purification process in order to obtain only one band meaning the protein is pure. Sample 1-Ladder used to approximate weight of the protein.Sample 2-E. coli (BL21) expressing pGEM-gbr22. Sample 3-lysed bacteria treated with benzonase in first steps of purification. Sample 4: flow-through of supernatant flushed with Ni-NTA resin. Sample 5-remaining proteins after being washed with Imidazole. Sample 5-first elution after using midazole most protein should be washed in this sample. Sample 6-second elution also after using Imidazole the waste should be here.The estimate the molecular weight of the protein is around 25 kDa. Lysozyme was added to the pellet suspension to break down the cell wall of the bacteria like Benzonase was added to to degrade the DNA and RNA. The HIS tag system works by attaching histidine to the C-terminus of the protein. (Histidine doesn't really "attach", it is just co-transcribed and translated) Histidine residues bind to nickel in order to be immobilized on a Ni-NTA column matrix. The main difference between wash buffer and elution buffer is that wash buffer contains less Imidazole and it removes loosely bound proteins while elution buffer contains a greater amount of Imidazole and it competes with histidine residues as well as causing the release of more proteins during flow-through. (Sources of Error?!)

__ Conclusions: __ ==== The key objective of the lab was to learn the process of the expression, purification and characterization of a protein which in this case was fluorescent purple. The creation of recombinant proteins, purification using an affinity tag and Ni-NTA resin, and characterization of the protein by using gel electrophoresis to determine the concentration. Key findings were discovering that the protein purification was not completely successful since it was only 50% pure due to the appearance of a second band. In addtion, the determined MW of the protein was ~ 25 kda which was not expected. Future applications of this experiment could be the expression, purification and characterization of our own target protein, using the same procedures in other proteins and further testing on this protein such as virtual drug screening it. ====

__ References: __ (Need one more?) 1. Nat Methods. **2008** Feb;5(2):135-46. Protein production and purification. 2. []