JESSICA+N

__Introduction: __
====Many studies have provided a consensus protocol for generating purified recombinant proteins [1]. The cDNA is obtained by amplifying either genomic DNA or full-length, sequence-verified cDNA’s. The ligation-independent cloning clones the full-length cDNA into an E.coli expression vector. The T7 RNa polymerase is used as a driven expression and an N-terminal oligohistidine tag. Thus, the protein is expressed as [in?]  a copy of the E.coliBL21(DE3) strain (the protein is expressed using E. coli) . The protein is solubilized and purified in a well-buffered solution, and the immobilized metal affinity chromatography is used as the initial purification step; gel filtration is used to increase purity of the protein sample. The affinity tag can be removed to minimize non-native sequences in the recombinant protein and to achieve further purification. A recombinant, hexahistidine-tagged protease removes the protease (a protease removes a protease?)  and any cellular proteins that bound to the metal affinity resin [1,2]. This lab is consisted of three parts: protein expression, purification and characterization. Thus, the purpose of this lab is to overexpress a recombinant protein in bacteria, to purify the overexpressed protein, and to analyze the samples using the gel electrophoresis. The purified protein goes through characterization to analyze the purity of the protein. The SDS-PAGE denatures proteins to discriminate different proteins in a mixture. The UV-Vis spectroscopy measures the concentration of the protein solution. If the protein pGEM-gbr22 is properly expressed, purified, and characterized, then the estimated molecular weight of the purified protein will be about 25 kDa. ====

__Materials & Methods: __
====25 μl of competent bacteria were added to the DNA and Control tubes. Plasmid was added to the DNA tube. The plates were heat shocked in the water bath. SOC was added to the two plates, and placed in the incubator. 50 μl of bacteria/SOC mixture were pipetted on each place and colirollers evenly spread the bacteria. The next day, the starter culture was set up to be grown in LB supplemented with ampicillin, hence large cultures were made for overnight expression. The purple culture was harvested and 500 μl of sample were dispensed into sample 1. Remaining purple culture was centrifuged and the purple pellet was saved. Cyanase was added to the lysed E.coli cells. The lysate was distributed into several tubes and centrifuged. After centrifugation, the supernatant was dispensed into a tube (sample 2). The liquid supernatant was isolated from the cell debris pellet. The lysate was syringe filtered. The protein was purified through Ni-NTA affinity purification: Ni-NTA resin/buffer mix was added to the column, discarding flow through (sample 3); the wash buffer was added to the column, discarding flow through (sample 4); the elution buffer was added to the column, retaining flow through (sample 5 and 6). The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to measure the reading for the protein solution at 280 nm and at maximum wavelength. 500 μl of water was pipetted into sample 1, was centrifuged, and pellet was retained, which was resuspended in water and 6x gel loading buffer was added. The loading buffer was added to samples 2-6. All sample tubes were heat blocked and then centrifuged. The electrophoresis was properly arranged and the samples were loaded inside. The gel was removed, washed and stained. 1-1.5 hours later, the gel was destained. The gel was dried for analysis. ====

__Results: __








Beer’s Law: A=Ebc Molecular weight of protein: 25794.2 g/mol

A= 0.31, E=38850 L/mol*cm, b=1cm c= (0.31)/[(38850 L/mol*cm)(1cm)] = (7.98 x 10-6 mol/L)(25794.2 g/mol) = __0.206 mg/ml (concentration)__
 * 280 nm **

Yield=cV (V=volume of elution) = (5.2ml)(0.206mg/ml) = __1.070mg yield__

A= (0.57+0.52)/2=0.545, E=118300 L/mol*cm, b=1cm c= (0.545)/[(118300 L/mol*cm)(1cm)] = (4.61 x 10-6 mol/L)(25794.2 g/mol) = __0.119 mg/ml (concentration)__
 * <span style="font-family: Georgia,serif;">574 nm **

<span style="font-family: Georgia,serif;">Yield=cV = (4.7ml)(0.119mg/ml) = __0.559 mg yield__

__<span style="font-family: Georgia,serif;">Discussion: __ <span style="font-family: Georgia,serif;">During the purification process, the bacterial cells had to release the soluble proteins and remove the insoluble cell debris, such as the cell wall and membranes. Lysozyme was used to break down the cell wall barriers of the bacteria, and Cyanase was used to reduce the viscosity of the mixture containing lysozyme by digesting the DNA/RNA in the mixture, thus removing the DNA from the protein. The overexpressed protein (gbr22) contained six histidine residues added to the C-terminus, which was utilized to separate the protein from other cellular proteins - the histidine residues bind to divalent cations, such as Ni-NTA agarose. When imidazole was added, the protein was released from the Ni-NTA agarose. The wash buffer removed proteins that were loosely bound to the resin, while the elution buffer released the protein from the Ni-NTA resin as it contained a higher concentration of imidazole <span style="color: #ff0000; font-family: Georgia,serif;">(what role does imidazole play in allowing the protein to flow through the column? Be specific) <span style="font-family: Georgia,serif;">. Six samples were made during protein expression and purification. Sample 1 contained LB media and ampicillin, sample 2 contained the mixture after lysing the cell, sample 3 was obtained after the resin and buffer flowed through the column, sample 4 contained the wash buffer – 1x PBS with 20mM of imidazole, sample 5 contained the elution buffer 1 – 1x PBS with 250mM of imidazole, and sample 6 consisted of the elution buffer 2 <span style="color: #ff0000; font-family: Georgia,serif;">(did any of these samples have protein in them? Make sure to include what the samples contain) <span style="font-family: Georgia,serif;">. <span style="font-family: Georgia,serif;">From the absorbance at 280 nm and 574 nm, the concentration of the protein came out to be 0.206mg/ml and 0.119mg/ml, and the yield turned out to be 1.070 mg and 0.559 mg, respectively. Using sample 5 as a reference, the size of the protein was about 25 kDa; thus, the results supported the hypothesis. However, in the sample 5 lane, another protein band with equal intensity was present, giving an estimated purity of the final protein of 50%. These errors may have occurred due to unsterile techniques and contamination of solutions.

__<span style="font-family: Georgia,serif;">Conclusions: __ <span style="font-family: Georgia,serif;">This lab was conducted to overexpress the recombinant protein pGEM-gbr22 in bacteria, to purify the protein by releasing soluble proteins and removing insoluble cell debris by lysing the E.coli cells and using the affinity tag and Ni-NTA resin, and to analyze the samples using the gel electrophoresis. A key finding is that the molecular weight of the final purified protein was 25 kDa, while the purity was 50%. In the future, this lab can help in drug discovery and determine if the proteins can be used as a drug target.

__<span style="font-family: Georgia,serif;">Ref __<span style="font-family: Georgia,serif;">erences: <span style="font-family: Georgia,serif;">[1] Structural Genomics Consortium, Protein production and purification. Nat Methods 2008, 5, (2), 135-46. <span style="font-family: Georgia,serif;">[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed Apr 15, 2013).