STELLA+M

Add more about this lab to intro

Missing error analysis

Isolation of Fluorescent // Montipora efflorescens // Protein gbr22 through E. Coli Expression

__ Introduction: __ Proteins expressed with recombinant DNA can use a variety of hosts, such as bacteria, yeast, and even mammalian cells. E. Coli was the first organism used to express a recombinant protein for pharmaceutical use in 1982, and was able to drastically reduce the cost of insulin.[1] Although an economical solution, more complex cells have started to become more popular for expression due to their ability to express more complex proteins.[1] Purification of recombinant proteins following the IMAC method, or immobilized metal affinity chromatography, is efficient because it has strong, specific binding.[2] In this experiment, E. Coli will be used as a host to express a recombinant HIS-tagged purple protein which will be purified using bead-bound nickel atoms in column chromatography. This purification will be able to be verified using an SDS PAGE gel.

__ Materials & Methods: __ E. Coli BL21(DE3) from New England Bio-labs was used as the expression organism. 2 µL of plasmid pGEM-gbr22 was added to 25µL of bacteria in one transformation tube, and 25µL of bacteria was added to a second transformation tube as a control. After 30 minutes on ice, both tubes were heat shocked for 45 seconds in a water bath at 42ºC. 200µL of SOC media was added to each tube, and the tubes were placed in the shaking incubator at 37ºC and 250rpm for 30 minutes. 50µL of bacteria/SOC mixture was added to 2 pre-warmed agar plates coated with ampicillin from the two transformation tubes. The bacterial plasmid pGEM-gbr22 contains a gene for ampicillin resistance, ensuring only successfully transformed BL21(DE3) bacteria appear on the experimental plate. Plates were stored in 37º incubator overnight. 10µL of 50mg/ml ampicillin was added to each of 2 sterile culture tubes with 5ml of LB. A bacterial culture containing pGEM-gbr22 was selected with a pipette tip and dropped into each solution. Tubes were incubated for 8 hours. 625µL of starter culture was added to an Erlenmeyer flask containing 25ml of LB and 50µL of 50mg/ml ampicillin and incubated for 24 hours. 500µL sample of culture was taken for PAGE analysis. Remaining culture was centrifuged at 5000 rpm for 10 minutes. Liquid was decanted and 2.5mL of 1xPBS and 51µL of 50µg/µL added. 2µ of cyanase was added to tube after reaching room temperature. The lysate was distributed into micro-centrifuge tubes and centrifuged. 50 µL of supernatant was kept for PAGE analysis. The rest of the liquid was transferred to a 15mL conical tube. Lysate was syringe filtered through .22µm syringe. 0.5 ml of Ni-NTA resin/buffer mix was added to this solution and transferred to a 20 ml Bio-Rad chromatography column after settling. Flow though was collected and 50µL retained for PAGE analysis. A wash buffer of 5ml of 1xPBS and 20mM imidazole was added and the flow-through collected, 50µL being retained for PAGE analysis. An elution buffer of 1xPBS and 250mM imidazole was added and the flow-through collected, and 50µL retained for analysis. This step was repeated and another 50µL collected. Absorption spectra were obtained for 1st elution using a Nanodrop spectrophotometer at 280nm and 574nm. The 6 samples taken for analysis were run through a PAGE gel using an SDS buffer. __ Results: __ Figure 1: Many E.Coli BL21(DE3) cultures expressing plasmid pGEM-gbr22 growing on ampicillin treated agar plate after overnight incubation.

Figure 2: Control plate showing no BL21(DE3) growth due to the ampicillin treated agar plate after overnight incubation

Figure 3: Large culture of BL21(DE3) showing over-expression of gbr22 purple protein after incubation

Figure 4: Wet pellet weight of .5 grams after centrifugation of large culture of BL21(DE3). The purple pellet contains protein gbr22.

Figure 5A (left): Slight purple tint indicates presence of gbr22 protein in first elution after purification steps Figure 5B(right): Clear solution of elution 2 indicates the absence of gbr22 after purification steps

Figure 6: Nanodrop spectrophotometer absorption at 280nm for elution 1 containing gbr22 showing an absorbance of .168

Using Beer's Law to calculate the yield from the absorbance: A=εBC, where A is absorption, ε is extinction coefficient, B is cuvette length, and C is concentration: A =.168 B = 1cm Extinction coefficient at 280 nm: 33850 M-1 cm-1 C = (0.168)/(38850) = 4.324E -6 M Molecular weight of gbr22: 25,794.2 g/mol

With final volume of 5ml: 4.324E-6 M (.005L)(25,794.2g/mol) = .558 mg gbr2 Figure 7: SDS PAGE gel results from samples 1-6 (lanes 2-7) from one series of purification steps and samples 4-6 (lanes 8-10) of another series of purification steps. Lane 9 contains the elution for which yield was calculated. Lane 1 contains standards for comparison.

Figure 8: SDS PAGE molecular weight standards from 10-170 kDA from Thermo Scientific

__ Discussion: __ The lysosyme added to the pellet breaks down the cell walls of the bacteria and releases the contents of the cell, including the gbr22 protein. The cyanase breaks down the bacterial DNA and RNA to facilitate further purification. This particular gbr22 protein is HIS-tagged, which means a chain of 6 histidine amino acids are added to the end of the protein. The histidines will bind to nickel atoms attached to a large bead present in the Ni-NTA resin. The large beads prevent the nickel atoms, and the protein bound to it, from falling through the chromatography column. Imidazole acts as a competitive inhibitor for these nickel atoms, and when a lot is added, the histidines will detach from the resin and they, and the protein attached it them, will fall through the column. The first sample taken for PAGE analysis was simply the lysed bacterial cells with all of their native proteins, indicated by the many bands for sample 1 in figure 7. Sample 2 was taken after the cyanse had broken down the DNA and RNA and the larger macromolecules were left in the pellet after centrifugation. Sample 3 was the waste after the supernatant had been added to the Ni-NTA resin and placed in the chromatography column. Sample 4 was collected after the wash buffer and been added to the column. The wash buffer contains a small concentration of imidazole, so proteins loosely bound to the Ni-NTA resin would fall through. At this point, the gbr22 protein should still be bound to the resin due to the HIS tag. Sample 5 was taken from elution 1 using the elution buffer, which contains a high concentration of imidazole that would remove the protein from the resin. This is the fully purified protein, and can be seen most clearly as the dark band in lane 9. Sample 6 was a second wash with the elution buffer, and since most of the protein was removed with the first elution, it is expected that no protein would appear on the PAGE analysis, which is consistent with lanes 7 and 10 in figure 7. The purity of the second is much higher than the first, at about 80% in lane 9 and 60% in lane 6 (both 1st elutions). This may be due to better purification technique or having an overall higher yield of protein from initial expression. The darkest band in both elution 1's appear just above 25kDa according to the standards in figure 8. This agrees with the known value of the molecular weight of 25,794.2 g/mol.

__ Conclusions: __ A plasmid containing a gene for a fluorescent protein was introduced to E-coli, over expressed, purified, and characterized. Using the methods outlined above to isolate and correctly identify the protein, as well as estimate the yield, experiments can then be performed using this protein. Specifically, ligands chosen by virtual screening programs can be tested for their ability to inhibit the protein obtained.

__ References: __

1. Swartz, J. R., Advances in // Escherichia coli // production of therapeutic proteins. // Current Opinion in Biotechnology //** 2001, ** // 12 // (2), 195-201.

2. Graslund, S.; et. al., Protein production and purification. // Nat. Methods //** 2008, ** // 5 // (2), 135-146