KATHERINE+V

__ Title: __
 * Expression, purification and characterization of gbr-22 protein **

__ Introduction: __ The bacteria //Escherichia coli// have been a common expression host for the production of various soluble proteins [1]. Recombinant proteins are created using recombinant DNA from molecular cloning [2]. These proteins are used all throughout biological and biomedical sciences [1]. This lab is divided into three subparts: protein expression, purification and characterization. BL21 (New England BioLabs, Ipswich, MA), a common strain of E. coli is used as the expression host and transformed by the plasmid pGEM gbr-22, which encodes for a fluorescent protein (This protein is not fluorecent it's just purple) that displays a purple color to allow it to be easily followed throughout the three processes. The plasmid also contains antibiotic-selection marker, ampicillin, for the identification of successfully transformed bacterial colonies. (explain how exactly this occurs) Protein purification consists of the addition of lysozyme to break down the cell membrane of the E. coli strain, and then the protein of interest is separated. The modified recombinant protein contains a C-terminal hexahistidine affinity tag (How exactly does this occur? what happens when imidazole is added?) and the proteins without the tag are disposed of. The purified protein then undergoes characterization to analyze the quality of the protein. SDS-PAGE denatures the protein and tests for the purity of the sample along with checking if the protein is of expected size [1]. UV-Vis spectroscopy is also used to identify the concentration of the protein. (please explain each step. Remember to address why we do each step and its significance.) If the protein pGEM gbr-22 is expressed, purified and charcterized, then the expected protein characterization will result in the molecular weight near 25 kDa. __ Materials & Methods: __ To express the recombinant protein, the bacterial cell E. coli BL21 (DE3) was transformed by the ampicillin-resistant expression plasmid pGEM gbr-22. The transformed bacteria were added to a transformation and control test tube, then heat shocked. To prevent contamination, This is not why SOC was added. SOC has extra nutrients in it so that the bacteria can recover from being heat shocked. SOC media was added and the sample were transferred to agar plates and left to incubate overnight at 37⁰C. After overnight growth, a starter culture was selected and placed in a flask containing ampicillin and nutrient rich LB, then incubated. The purple protein, sample 1, was harvested using the Allegra X-15 Centrifuge (Beckman Coulter Inc., Brea, CA). 1x PBS and lysozyme was then added to the purple pellet and stored at -20⁰C. Afterwards, Benzonase was added to the conical tube containing the lysate and centrifuged. The supernatant was saved as sample 2. Then the lysate was syringed filter to remove large particulate matter and purify the protein. Ni-NTA resin/buffer was mixed with the protein to be used in a Bio-Rad chromatography Econo column. The protein was isolated with 1x PBS and 250mM Imidazole for samples 2-6. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the concentration of sample 5 proteins. For protein characterization, an electrophoresis module – Mini-PROTEAN tank – was assembled and filled with 1x TGS buffer. The samples – ladder, LB flask, soluble proteins, flow through, wash, elution 1, elution 2, and partner’s wash, elution 1, elution 2 - were loaded into the wells respectively. A gel was produced, stained and dried for analysis of gbr-22 protein. You can either explain each step here or in the introduction. This would be a good M&M if you explained why we did each step in either the introduction or here. __ Results: __

















//** CAPTIONS???????!?!?!??!!?!??!!!!!!!!?!? **// Beer's Law: A = Ebc Molecular weight of gbr-22 = 25,794.2 g/mol

A = 0.33, E = 38,850 L/mol*cm, b = 1 cm c = A/Eb = (0.33)/(38350 L/mol*cm)(1 cm) = (8.57 x 10-6 mol/L)(25794.2 g/mol)= **__0.221 mg/ml concentration__**
 * 280 nm **

Yield = cV = (5ml)(0.221 mg/ml) = **__1.105 mg yield__**

Average absorbance of 2 trials: (0.59 + 0.52)/2 = .555 A = 0.555, E = 118300 L/mol*cm, b = 1 cm c = A/Eb = (0.555)/(118300 L/mol*cm)(1 cm) = (4.69 x 10-6 mol/L)(25794.2 g/mol) = **__0.121 mg/ml concentration__**
 * 574 nm **

Yield = cV = (5ml)(0.121 mg/ml) = **__0.605 mg yield__** Good job with the calculations!





__ Discussion: __ At 280 nm and 574 nm, the protein concentration was calculated to be 0.221 mg/ml and 0.121 mg/ml, respectively. The purified protein yield was 1.105 mg at 280 nm and 0.605 mg at 574 nm. The difference in concentration and yield can be accounted for due to the different wavelengths. Aromatic amino acids – tyrosine, tryptophan and phenylalanine – absorbs at 280 nm and results in higher values because the Nanodrop spectrophotometer quantifies these amino acids as part of the protein, when in fact they are not (explain this a bit. it's not completely clear). While the hypothesis was correct because the gel depicted the molecular weight of gbr-22 to be around 25 kDa in lane 7, there was another protein band of equal intensity present at approximately 18 kDa. With another protein present, it can be concluded that the protein was only 50% pure. This could have occurred due to contamination of equipment or solutions, inaccurate readings or incorrect sterile techniques. The purification process involved various compounds to isolate the expressed protein. Lysozyme was used to break down the cell walls of the bacteria, while Cyanase was used to remove nuclei acids or DNA from the protein solution. The gbr-22 protein contained a hexhistinide tag to the C-terminus allowing it to bind to the Ni-NTA agarose. To extract the wanted protein, a Wash solution with a small concentration of imidazole removed most cellular proteins while an Elution solution with a stronger concentration of imidazole eluted the tagged protein. Six samples were taken to view the steps in the expression and purification process. Sample 1 was taken from the flask containing LB media and ampicillin. Sample 2 was obtained after lysing the cell and syringe filtering. After the buffer/resin mix flowed through the column, sample 3 was obtained. The wash step with 20mM imidazole and 1x PBS provided sample 4. Sample 5 consisted of gbr-22 after an elution buffer of 250mM imidazole and 1x PBS was poured into the chromatography column. Finally, the elution buffer was added again to create sample 6. You should put some of this in your intro and in your captions. __ Conclusions: __ The purpose of the lab was to introduce the process of protein expression, purification and characterization. This was done by expressing the fluorescent coral protein gbr-22 in BL21 (DE3) E. coli bacteria, purifying by lysing the cell and removing insoluble proteins, isolating the protein with Ni-NTA resin/buffer and an affinity tag, and characterizing the protein with SDS-PAGE. The final protein had a molecular weight of 25 kDa, but had a purity of 50% because there was another protein present at approximately 18 kDa. This lab is critical because the processes will be used again for future studies of target protein and drug discoveries. (this is good but it can be improved..)

__ References: __
 * 1) Structural Genomics Consortium. Protein production and purification. Nat Methods. 2008;5:135–146. et al.
 * 2) Protein Expression and Purification Core Facility. [] (accessed April 16).