MANUEL+Z

__ Title: __ Isolation of gbr22 through expression and purifcation followed by characterization through gel electrophoresis

__ Introduction: __ Protein expression is a useful technique widely used for research in various fields of study and medicine. There was once a time when recombinant proteins were only produced by experts, but present day availability of simpler systems of protein expression has made the practice widespread. Protein expression can be carried out in a number of different cells include those bacteria, yeast, insects, and even humans. Proteins that have been expressed must then be isolated through a series of purification techniques, such as clarification and chromatography. In order to confirm successful isolation, the resulting purified samples may be characterized through another set of different techniques like the gel electrophoresis used in this lab. [1] In this series of labs, E. coli was transformed using a plasmid containing an encoding sequence for the purple fluorescent protein, gbr22. The bacteria was cultivated to overexpression of the recombinant protein and harvested. The bacteria cells collected were lysed and the purple protein was isolated through clarification and chromatography, ultimately collected in elutions which were analyzed with a Nanodrop spectrophotometer. The measurements made on the light absorbance of these elutions were implemented in the Beer’s Law formula, A = εbc, where A is absorbance, ε is molar absorptivity, b is the path length, and c is the concentration. The implementation of this data and formula allowed for determination of the final concentration of recombinant protein the collected elutions, which in turn allowed for derivation of the mass collected. Throughout the labs, samples were collected at various points in the expression and purification process and from the final elutions. Gel electrophoresis was then used to compare the contents of each of the samples and most importantly, characterize the protein contained within the final elutions. After the protein is expressed and purified, the elution in which it has been isolated will appear purple due to the purple fluorescence of the recombinant protein and the gel lane in which it was inserted should have only one line, indicating the approximate size of the protein and serving as confirmation of the successful purification of the desired protein.

__ Materials & Methods: __ The first part of the procedure was the expression of the protein. E. Coli cells were split up into to two transformation tubes. The plasmid pGEM-gbr22 was added to one. Both tubes were heat shocked and put on ice. SOC media was added and the tubes were shook in an incubator for 30 minutes. Colirollers were used to spread the contents on two separate plates. The plates were stored in a 37oC incubator overnight. The next day, cells were harvested and added to a tube containing LB broth and ampicillin. The tube was shook in the incubator for ~8 hours and then emptied into an Erlenmeyer flask containing LB and ampicillin which was capped with foil and placed in the incubator overnight. A 50mL sample of the contents was centrifuged in a conical tube to separate the pellet which was suspended in 1X PBS and lysozyme, mixed, and stored in a -20oC freezer. The next part involved purification. 2ml of benzonase was added to the thawed lysed sample, which was then clarified to remove the insoluble components. The supernatant was syringe filtered and added to a column containing Ni-NTA resin. Chromatography was executed using a wash buffer with 20mM imidazole and an elution buffer with 250 Mm imidazole. The absorbance of the obtained elution 1 was measured using a nanodrop spectrophotometer at wavelengths of 280nm and 574 nm. In the final step (characterization), gel electrophoresis was executed using 6 samples collected throughout the process. *gas burners used to sterilize area

__ Results: __

Figure 1: LB + Ampicillin media plate with ~350 colonies of BL21(DE3) bacteria transformed by the plasmid pGEM-gbr22

Figure 2: LB + Ampicillin media plate with ~0 colonies of non-transformed BL21(DE3) bacteria

Figure 3: Erlenmeyer flask with large culture of BL21(DE3) bacteria transformed with the plasmid pGEM-gbr22; purple coloration indicates bacteria growth

Figure 4: Conical tube with pGEM-gbr22 transformed BL21(DE3) bacteria pellet; purple color indicates the presence of the desired purple protein; pellet weighed ~.343g

Figure 4: Conical tube with pGEM-gbr22 transformed BL21(DE3) bacteria pellet; purple color indicates the presence of the desired purple protein; pellet weighed ~.343g

Figure 6: Elution 2 from the second elution buffer flow through used in the column containing Ni-NTA; Elution 2 possibly has some purple protein left over after elution 1 was collected

Figure 7: Absorbance spectra generated by ND 1000 in the Protein A-280 setting; Absorbance for trial 1 at 280nm is 0.294

Figure 8: Absorbance spectra generated by ND 1000 in the Protein A-280 setting; Absorbance for trial 2 at 280nm is 0.262

A = ԑbc à  c = A/ԑb

Concentration at 280 nm: Aavg=.278 c= 0.278/(38850L*M-1*cm-1 X 1 cm)= 7.1557*10^-6 mol/L X (25,794.2 g/mol) = 0.1835 g/L or mg/mL

Concentration at 574 nm: Aavg=.46 c= 0.46/(118300L*M-1*cm-1 X 1 cm)= 3.888*10^-6 mol/L X (25,794.2 g/mol) = 0.1003 g/L or mg/mL

Figure 9: Dry electrophoresis gel; PageRule Prestained Protein Ladder in far left column and samples 1-6 follow to the right

Figure 10: Moleculer weight standard used in gel electrophoresis; PageRule Prestained Protein Ladder #26616

__ Discussion: __ Expression, purification, and characterization of the fluorescent purple protein gbr22 were all carried out in this series of procedures. Characterization was accomplished with the aid of plasmid transformed bacteria. The bacteria served as a means of amplification. The plasmid also induced resistance to ampicillin in the transformed bacteria. The use of ampicillin ensured that only transformed bacteria would be used in the procedures. Once the transformed bacteria were harvested within the purple pellet, lysozyme was used to digest the cell walls and benzonase was used to digest DNA and RNA of the bacteria. Clarification was used to isolate the proteins, now removed from the rest of the bacterial cells. Column chromatography was then used to wash out any unwanted proteins from the remaining sample. The column used contained Ni-NTA resin. The purple protein would bind to the Ni within the resin because of HIS tag introduced by the initial plasmid (pGEM-gbr22). The HIS tag contains 6 histidine residues in a row (an uncommon occurrence in nature) which each have a group that readily binds to the Ni in the resin. The wash buffer used to wash out unwanted proteins (which at most weakly bind with the resin) contains a small concentration of imidazole. The elution buffer used to remove the purple protein from the resin contained a much higher concentration of imidazole. Imidazole is actually identical to the group within histidine that allows it to bind to the Ni. At higher concentrations of imidazole, the purple protein is less likely to bind to the resin and therefore falls through the column opening and into the elutions collected. The nanodrop readings on elution translated to a concentration of 0.1835 mg/mL (at 280nm) and 0.1003 mg/mL (at 574 nm). With the volume of elution 1 estimated to be 4.55 mL, the calculated percent yields were 0.8349 mg and 0.4564 mg. Once the protein was purified, characterization was executed in order to confirm the identity of the protein obtained. Gel electrophoresis was used with 6 samples collected throughout the entire process. Sample 1 contained the lysed transformed bacteria (with purple protein inside), sample 2 contained the supernatant after centrifugation of the lysed cells, sample 3 contained proteins that did not bind to the Ni-NTA at all, sample 4 contained proteins that were washed out with the wash buffer, sample 5 contained the eluted gbr22 protein in elution 1, and sample 6 contained the eluted gbr22 protein in elution 2. When the gel electrophoresis was completed, samples 5 and 6 were examined for bands. Both 5 and 6 had 1 prominent band, indicating high purity. Sample 5 only exhibited minor presence of contamination, with purity estimated around 80%. Using the molecular weight standard shown by the prestained ladder in lane 1 of the gel, it was estimated that the molecular weight of the purified protein is ~25 kDa. Error may have been introduced throughout the experiment by failure of equipment, contamination of material and samples, sloppy technique, and error in calculation, but the experiments were each executed steadily and without much trouble.

__ Conclusions: __

The isolation of the protein gbr22 was executed successfully in this series of procedures involving expression, purification, and characterization. Using a nanodrop spectrophotometer, the concentration of purple protein in the elution contained was about 0.1001 mg/mL. This concentration is relatively low and indicates a low yield. However, characterization using gel electrophoresis indicated little to no presence of unwanted contaminants in the purified elution of gbr22. Future directions include utilization of the isolated protein for drug assays or isolation of a different kind of protein through the same or different means. The experience gained through this protein isolation process will prove instrumental in future procedures in virtual drug screening.

__ References: __

[1] Acton, T.B.; Albeck, S.; Almo, S. C.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. Nat Methods 2008, 5, (2), 135.