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 * __ Protein Production and Purification __**

**__ Introduction: __** The stably folded, globular domains of prokaryotic and eukaryotic proteins are a major focus of the biomedical research community. These proteins are generally suitable for expression in //E. coli//. Over the years, much effort has been put into optimizing E. coli as an expression host for proteins from higher organisms. This strategy has generated a wide arsenal of tools that can be used to increase the yield of soluble protein. 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//. 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. For high-level protein production purposes, BL21(DE3) is an appropriate E. coli strain. Historically, ampicillin has been the most commonly used antibiotic-selection marker. The expression plasmid (pGEM-gbr22) carries a gene for ampicillin resistance and the fluorescent protein (that the plasmid encodes for) has six histidine residues appended at the C-terminus. The hexa-histidine tag is an affinity tag that allows for fast and efficient purification of the protein. Most purification steps can be integrated by high-performance liquid chromatography. Various factors that can affect the result of a protein purification experiment include: lysis method and conditions, buffer composition, temperature, protein solubility. 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. Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality. If size exclusion chromatography was used as the last purification step, a close look at the chromatogram through gel filtration is essential. It is also of value to analyze the protein in each peak by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) or mass spectrometry. Through these various methods, a recombinant protein in //Escherichia coli// bacteria are overexpressed, purified and characterized in order to analyze and study human proteins without having to use human tissues.

**__ 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, 10x PBS, 1x PBS, 1.7 ml centrifuge tubes, 2 x 10 ml round bottom tubes, 4 x15 ml conical tubes, Bio-rad Econo chromatography column with yellow cap and clear round top, Ring stand and clamps, Ni-NTA resin and Benzonase – keep on ice · __ Protein Characterization: __ Mini-PROTEAN electrophoresis tank and lid, Power supply and leads, TGS running buffer, Bio-Rad precast polyacrylamide gel, 100 m l 6x gel (or sample) loading buffer, Protein Samples #1-6 from Expression and Purification labs, Molecular Weight standards, Plastic container w/ lid, Imperial protein stain, Whatman paper, sarah wrap, gel, and labeling pen.

**__ Methods: __** __ Protein Expression: __ 1. Day 1 – Transforming the competent bacterial cells. Ø We had 3 plates: __ one experimental plate with DNA __ and __one Control plate__ that had no DNA to confirm that we had a clean technique and no colonies were growing without the plasmid. We also had __one plate for ‘fun’__ – sneezed on; since it did not have any antibiotic mixed in the agar – microorganisms such as yeast, bacteria and mold were able to grow. Labeled with the type of __antibiotic__ (e.g. Amp or None), name of the organism (e.g. __BL21(DE3)__, vector name (e.g. __pGEM-gbr22__ or __No DNA control__), the __date__, __VDS__, and __your initials.__ Used gas burner to sterilize general area when handling the bacteria.  2. Day 2 (morning) - Growing a starter culture.   Ø Grew starter protein in LB supplemented with 100 m g/ml ampicillin.  3. Day 2 (Evening): Protein expression in large culture.   Ø Made the large culture for overnight overexpression in an incubator operating at 37oC and 200-350 rpm.  4. Day 3 or 4 - Harvesting cells. Ø Harvested the cells when the culture was purple using the centrifuge with temperature set at 4oC. Afterwards, resuspended the cells in phosphate buffered saline.

__ Protein Purification: __ 1. Lysed the //E. coli// cells using lysozyme. 2. Clarified the lysate. After the centrifuge, took a 50 m l sample of the supernatant and labeled it sample 2. 3. Isolated the soluble fraction. 4. Syringe filtered the lysate. 5. Prepared the Wash and Elution (1&2) buffers using 1x PBS with Imidazole. 6. Purified the protein using a combination batch (Ni-NTA resin/buffer) and column chromatography. Took a 50 m l sample of the flow through and labeled it sample 3 and 50 m l sample of the wash step and labeled it sample 4. Also, took 50 m l sample of the each Elution 1 and Elution 2 and labeled them sample 5 and sample 6 respectively. 7. Using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) to measure the concentration of our final purified protein at 280nm wavelength and at the maximal wavelength, 574nm.

__ Protein Characterization: __ 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: Ø Dried the gel using a drying gel with the temp at 75oC, and gradient cycle for 1.5 hrs.

**__ Results: __**

** Protein Expression Lab  ** Figure 1: The bacterial colonies in our DNA plate, no DNA plate and fun plate. Nothing grew in out plates so there are no purple marks.

Figure 2: Purple culture in the flask; The purple means that bacteria grew.

Figure 3: The cell pellet from the purple culture; Pellet A mass: 0.67 grams; Pellet B mass: 0.94 grams

** Protein Purification Lab ** Figure 1: Elution 1 (a little purple) and Elution 2 (clear) of purified protein; We had to use another group's elution 1 concentration from absorbance at 280nm and at maximal wavelength because our concentrations were negative.

Figure 2a: Nanodrop measuring absorbance at 280 nm of Elution 1 using Protein A280 mode at the first trial with a yield of -0.04 mg/ml.

Figure 2b: Nanodrop measuring absorbance at 280 nm of Elution 1 using Protein A280 mode at the 2nd trial with a yield of -0.02mg/ml.

Figure 3a: Nanodrop measuring absorbance at the maximal wavelength (574 nm) of Elution 1 using UV/VIS mode at the first trial.

Figure 3b: Nanodrop measuring absorbance at the maximal wavelength (574 nm) of Elution 1 using UV/VIS mode at the 2nd trial.

Ø Determining the concentration of the protein in mg/ml using Beer’s Law from the absorbance measured at wavelength 280nm: o Absorbance = [(0.079 + 0.122)/2] = 0.1005 o Extinction Coefficient = 38850 g/l = 38850 mg/ml § A = Ebc C = A/Eb = [(0.1005)/ (38850mg/ml)(1ml)] = 2.587 x 10-6 mg/ml Ø Determining the concentration of the protein in mg/ml using Beer’s Law from the absorbance measured at maximal wavelength 574nm: o Absorbance = [(0.097 + 0.098)/2]*10 = 0.975 o Extinction Coefficient = 118300 g/l = 118300 mg/ml § A = Ebc C = A/Eb = [(0.975)/ (118300mg/ml)(1ml)] = 8.24 x 10-6 mg/ml

** Protein Characterization Lab **

Figure 1: Molecular weight standard of PageRuler Prestained Protein Ladder (brand: Fermentas, catalog #: SM0671) between 10-170 kDA

Figure 2: A preliminary picture of the gel after the overnight wash and before drying it in case it didn't survive the drying process.

Figure 3: Our dried protein gel with the pageRuler prestained protein ladder (brand: Fermentas, catalog #: SM0671) in lane 1, sample 2 in lane 2, sample 3 in lane 3, sample 4 in lane 4, sample 5 in lane 5 and sample 6 in lane 6.

**__ Discussion: __** In our protein expression lab, our bacteria culture didn’t grow in our plate with the DNA, no DNA and fun plate because they didn’t contain any purple marks (as seen in Figure 1 under protein expression lab) so we had to use another group’s bacteria to make the large culture for the overnight expression. Afterwards, it was grown enough for us to harvest the cells as shown in Figure 2 (under protein expression) with the purple media and Figure 3 (under protein expression). In our protein purification lab, the concentration of our protein using maximal wavelength at 574nm was slightly different from the concentration found in when the absorption at 280nm wavelength was used; the yield if 280nm wavelength was used was 1.16415 x 10-5 mg while at the maximal wavelength (574nm) the yield was 3.708 x 10-5 mg. Using nanodrop spectrophotometer, our concentrations at the 280nm wavelength and the maximal wavelength 574nm using Elution1 was negative so we had to use another group’s Elution 1 to measure the absorbance at 280nm and 574nm and determine their concentrations. In our protein characterization lab, we prepared SDS-PAGE gel samples and dried the gel after it was washed overnight as could be seen in Figure 2 and Figure 3 under the protein characterization lab. Using the molecular weight standard (as shown in Figure 1 under the protein characterization lab) we were able to estimate the molecular weight of our purified protein. The imperial protein stain in lane 1 had a lot of bands because it had the cell lysate; bands for all proteins. Sample 2 in lane 2 had fewer bands than lane 1 (but still a lot of bands) because it contained the soluble plus pellet (after centrifugation). Sample 3 in lane 3 had few bands because it contained the flow through. Sample 4 in lane 4 barely had any bands because it contained the wash (20mM imidazole). Sample 5 in lane 5 had no bands because it contained Elution 1 (250mM imidazole); had no high concentration of protein. Like sample 5, sample 6 in lane 6 had no bands because it contained Elution 2 which had no high concentration of protein.

**__ Conclusion: __** In the protein expression lab, we overexpressed a recombinant protein in //E. coli// bacteria by growing the //E. coli// cultures to express the purple protein, harvest the cells by centrifugation and resuspending them in a phosphate buffered saline and freezing them (in -20oC) in order to purify them in the next lab. In the purification lab, we purified our protein overexpressed in bacteria from the previous lab. We lysed the bacterial cell debris by centrifugation and purified it using the affinity tag and Ni-NTA resin. In the protein characterization lab, we analyzed our purified protein (in samples 2-6) from the previous lab using gel electrophoresis. Using SDS-PAGE (sodium dodecyl sulfate polyacrylamide – used to separate proteins in our samples) and the spectroscopy results, we estimated the molecular weight of the gbr22 protein as well as the purity and yield of the final purified protein product.

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