MOHAMMAD+Q+A

=__ Title: __ From Bacteria to Protein: The Complicated Journey=

__ **Introduction**: __

Recombinant proteins have become increasingly important in science in recent years due to their importance and modifications in the fields of plant biology, vaccinations and treatment of diseases such as sickle cell anemia [1]. Recombinant protein is a general name for a protein that has been produced from recombinant DNA that has been made from combining many different pieces of DNA [1]. Being able to produce recombinant proteins using a host cell such as bacteria allows scientists to determine how the protein of choice would respond to different inhibitors, ligands or potential drugs and goes a long way in determining the efficacy of certain compounds against different types of diseases [2]. The objective of this lab was to perform the different steps in creating a recombinant protein such as the processes of protein expression //, // purification and characterization. Each step has its own role in cloning the DNA to insert into a bacteria (in this case- // Escherichia Coli // BL21 DE3), isolating it and then checking whether the protein has been purified successfully and contains no contaminants so that it may be used in experiments. The hypothesis is that if the protein was able to be expressed and purified correctly, then during characterization a band will appear near the 25 kDa (the reference molecular weight of the protein) mark during characterization.

Remember to state the name of the protein. You can also mention the fact that it codes for purple pigment in coral. Good intro and good job mentioning recombinant DNA.
 * __ Materials & Methods __**__ : __

First, the gene was cloned and inserted into pGEM-gbr22 plasmid. 25uL of bacteria were added to 2 transformation tubes and 1uL of plasmid DNA was added to the DNA tube but not the control tube, the samples were then placed on ice for 30 minutes, heat shocked at 42 ° C for 45 seconds in a water bath, 200uL of SOC media was added and the sample was shaken in an incubator for 30 minutes at 37 ° C and 250 rpm. Then, 50uL of the mixture were transferred to different plates and spread using colirollers to ensure even distribution. The plates were then stored in a 37 ° C incubator overnight. The next day, ampicillin and a sample of bacteria were added to two tubes of 5 mL LB broth. Then they were placed in the shaking incubator for 8 hours at 37 ° C at 200 rpm. After this, 25mL of LB broth and ampicillin were transferred to 2 erlenmeyer flasks, .625mL starter culture added and the mixture placed in the shaking incubator at 37 ° C and 200 rpm for one day. The bacteria were poured into a 50 mL conical tube and the Allegra X-15 centrifuge was set to 4 ° C for 10 minutes at 5000 rpm. The pellet and 50 uL of the liquid (Sample 1) was saved. The cells were suspended by preparing a 1x PBS solution, adding 2.5mL to the conical tube, vortexing, adding lysozyme to a concentration of 1mg/mL (stock: 50ug/ul) and placing the sample in a -20 ° C freezer. It was thawed, incubated at room temperature for 20 minutes, 1uL of Cyanase added, distributed into microcentrifuge tubes and centrifuged for 20 minutes at 14,000 rpm at 4 ° C and 50uL of this was saved as sample 2. Then, filtered through a .22 PES 5mL syringe filter, .5mL of Ni-NTA was added and was incubated at room temperature for 20 minutes. Mixture was added to 20mL Bio-Rad chromatography Econo column and run through with a wash buffer, elution buffer and then a stronger elution buffer. Samples 3-6 (50uL each) were collected after these steps. A nanodrop spectrophotometer was used at 280 nm to evaluate gbr22 protein absorbance in Elution 1. Sample 1 was microcentrifuged 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 95 ° C for 5 min and centrifuged again for 2 min at 5000 rpm. 7 uL of the MW standard (Thermo Scientific, Waltham, Massachusetts) was added to the first well with samples 1-6 being added in the subsequent wells and the electrophoresis procedure was run for 25 minutes [3]. The gel was then stained with Imperial protein stain, washed and dried at 75 ° C for 1.5 hours. This is an excellent summary of the proceedure and it smoothly transitions between steps. However, it is a good idea to explain some of the steps. For example why was lysozyme and cyanase used? What did Ni-NTA and Imidazole bind to? __ **Results**: __





















CAPTIONS?????????

Calculations:

Beer's Law A = εcb .72= 38850(c)(1) c= 1.85*10^-5 mol/L * 25794.2 (MW [3]) c= .477 g/L

**Discussion**: The ultimate result of this process was the gel that is shown in Figure 9. Although the gel was able to produce distinguishable, stained results, the experiment is not considered a complete success because in lane 6, there is more than one protein band. This is relevant because sample 5 is supposed to be the purest form of the gbr22 protein and should have only one protein band, indicating near perfect protein purity. Because there are two bands in the lane, it is assumed that the protein was contaminated at some point and there is only about 50% protein purity. The number of bands in the others do not matter because Well 1 contained the protein ladder, well 2 had just the cell lysate well 3 had soluble fraction of protein, well 4 had waste products such as lysozyme, well 5 had the Wash buffer and well 7 had the protein solution and Elution 2. Well 5 was relevant because it contained the Wash buffer. The wash buffer was added after first lysozyme and then cyanase were used to rupture the cell wall to expose proteins and to separate the DNA into manageable pieces so that they easily flowed through. In addition, a nickel ion resin attached to a large bead was used in conjunction with a 6x HIS tag to capture the gbr22 protein to prevent it from washing through when the Wash buffer was flowed through, thus ridding the solution of unwanted proteins Well 1 was relevant because it was the ladder used to distinguish the approximate weight of the protein. Comparing Figures 9 and 10, one can estimate the molecular weight of the protein to be around 25 kDa.

The difference between the wash buffer and the elution buffers was that the elution buffers had a much higher concentration of Imidazole (250mM) than the Wash Buffer (20mM) which caused more proteins to be released in the flow-through. This is very good, however a little more detail and explanation is needed. **Conclusions**: In this experiment, a protein was overexpressed in bacteria, purified and characterized in a gel to determine purity. Key findings included the fact that the protein purity was only about 50% and the molecular weight of the protein as determined by the electrophoresis procedure was about 25kDa. (put some of this in your intro as well)

Future applications of this experiment include purifying the protein further, performing enzyme assays and then using it to determine which ligands screened from virtual labs would make good inhibitors or drugs for the protein.

**References**: 1. Rensselaer Polytechnic Institute Department of Biochemical Engineering. The Basics of Recombinant DNA. [] (accessed Apr 17, 2013). 2. Protein production and purification.Nat Methods. 2008 Feb; 5(2):135-46.

3. ExPASy. ProtPram Lab. []