Ruifei+W

pGEM-gbr22 Protein Expression, Purification and Characterization through //E.Coli//

__ Introduction: __

====The process of obtaining protein for use in biochemical assays or antibody production involve protein expression and purification techniques [1]. The DNA coding for the protein must first be incorporated into a host cell which can range from bacterial to yeast cells. When using BL-21 bacterial //E. Coli//, a single recombinant colony is grown into a starter culture which is induced into protein expression and finally harvested for the target protein. Expression may be optimized by use of a tag (e.g. hexa-histidine affinity tag) or by varying expression induction conditions such as temperature and concentration [2] ==== ====The goal of protein purification is to isolate the protein. A clarified sample of soluble protein is first extracted via centrifuge and then clarified further by removing DNA and lipids. For His6-tags, a combination of imidazole buffer, phosphate buffer, and Ni-NTA resin is then used to bind and extract the protein [3].The success of expression and purification then can be gauged via gel electrophoresis and UV-vis spectroscopy. ==== ====Each individual protein has unique expression and purification protocols [1]. In this lab, gbr22, a purple protein found in coral, is expressed, purified, and characterized to determine the extent of purification. By utilizing appropriate expression and purification technique, characterization through gel electrophoresis should yield a molecular weight of gbr22 similar to the literature value as well as a high concentration of purified protein. ====

__ Materials & Methods: __

====gbr22 protein in the form of a plasmid with antibiotic resistance was mixed with a culture of bacteria and heat shocked to initiate transformation. The bacteria culture was then spread onto antibiotic plates incubated overnight at 37 O C to yield purple colonies of living transformed bacteria. A starter culture was then grown with a single purple colony, LB media, and 100 m g/ml ampicillin and put into a shake incubator at 37 O C for ~8 hours. The starter culture was then transferred into a larger culture with additional ampicillin and LB and shake incubated for 24 hours (sample 1 taken of large culture). The purple culture was then spun down to obtain a purple pellet of transformed cells. 1X PBS and 1 mg/mL lysozyme were added to the pellet and the final tube was frozen at -20OC. ==== ====The lysate was purified by benzonase to digest DNA, centrifuge (supernatant was taken as sample 2 at this point), and finally PES syringe filter of the clarified liquid lysate. The mixture was further purified via Ni-NTA affinity through a Bio-rad Econo column. 1X PBS, 20 mM imidazole, and 250 mM imidazole was run through the column to produce waste, wash, and two elutions. Samples 3, 4, 5, and 6 were taken of these steps, respectively. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of the first elution at 280 nm and 574 nm (the maximum absorbance wavelength of gbr-22). ==== ====In order to characterize the purified protein, gel electrophoresis was conducted with SDS-PAGE gel along with six samples taken at various points in the expression and purification steps (specified in the results section). The gel results were then compared to a molecular weight standard. ====

__ Results: __











__Calculation of Elution 1 yield based on Nanodrop results: __

Average absorbance values for n=2 trials = 0.538
 * 280 nm: **

Beer’s Law: A = abc, a = 38850 M-1cm-1, b = 1 cm, A = 0 .538

<span style="font-family: 'Times New Roman','serif';">c = 1.38 x 10-5 M (25794.2 gmol-1) = 0.36 mg/mL.

<span style="font-family: 'Times New Roman','serif';">yield280 = 4.5 mL * 0.36 mg/mL = **1.606 mg.**

<span style="font-family: 'Times New Roman','serif';">Average absorbance values for n=2 trials = 0.110
 * <span style="font-family: 'Times New Roman','serif';">574 nm: **

<span style="font-family: 'Times New Roman','serif';">Beer’s Law: A = abc, a = 118300 M-1cm-1, b = 1 mm, A = 0.110

<span style="font-family: 'Times New Roman','serif';">c = 9.30 x 10-6 M (25794.2 gmol-1) = 0.24 mg/mL.

<span style="font-family: 'Times New Roman','serif';">yield280 = 4.5 mL * 0.24 mg/mL = **1.080 mg.**

__ Discussion: __

<span style="font-family: 'Times New Roman','serif';"> The purification process involved the use of lysozyme to digest the bacterial cell walls and benzonase to digest the DNA and RNA present in the lysate. Then, the HIS affinity tag system, which was incorporated into the fluorescent protein, served to bind the gbr22 to the nickel ions in the Ni-NTA Sample 1 contained the bacterial cell sample suspended in LB media and ampicillin, sample 2 contained lysed cell parts excluding the protein, sample 3 contained 1 M PBS, sample 4 contained 20 mM imidazole, 1 M PBS, and loosely bound proteins to the Ni-NTA resin, sample 5 contained 200 mM imidazole, 1M PBS, and a majority of the gbr22 protein, and sample 6 contained 200 mM imidazole, 1 M PBS, and the leftovers of the gbr22 protein. Sample 4 contained wash buffer (20 mM imidazole, 1 M PBS) which served to remove proteins that did not bind to the Ni-NTA. Sample 5 contained elution buffer (200 mM imidazole, 1 M PBS) which served to remove the Ni-NTA bound protein – gbr22. <span style="font-family: 'Times New Roman','serif';"> The relevant gel lanes in figure 7 are those that contained sample 5 because it theoretically contained the majority of the purified protein. S5* was well-purified because there is one dark band corresponding to the gbr22 protein (with a molecular weight of around 26 kDa according to figure 8) and only a few lighter bands. S5* has a purity of around 80%. S5 also contained a dark band at around 26 kDa, but it was not purified as effectively because it contained two other dark bands and least two lighter bands. S5 has a purity of around 30%. This suggests that there was either error in preparing the gel lane or in the column elution steps. Overall, though the empirically determined MW of gbr22 in both S5 and S5* are fairly accurate with regards to the accepted value of 25.8 kDa. <span style="font-family: 'Times New Roman','serif';">The yield results based on the Nanodrop spectroscopy were fairly precise, with possible error in using the instrument due to contamination of the sample or a nonhomogenous sample.

__ Conclusions: __

<span style="font-family: 'Times New Roman','serif';">In this lab, gbr22 protein was expressed in //E. Coli//, purified, and characterized to determine the effectiveness of the expression and purification technique. It was found that the empirical technique was successful in extracting the gbr22 protein, but also had the potential of leaving unwanted proteins in the final sample. In addition, a yield on the order of 1 mg of protein could be obtained for a given colony of bacteria. <span style="font-family: 'Times New Roman','serif';"> In future VDS research, the process of utilizing bacteria to accurately harvest proteins is integral. Drug ligand candidates are very specific to which proteins they bind to, so it is important that the target protein be isolated in the presence of the ligand, else misleading results may be obtained. Knowing the procedure and being able to modify it at different steps is also important.

__ References: __

=
[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility: Protein Expression. [] (accessed April 15 2012). ======

=
[3] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility: Protein Expression. http://www.embl.de/pepcore/pepcore_services/protein_purification/index.html (accessed April 15 2012). ======