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//** Bacterial Protein - (Expression, Purification & Characterization) **//
 * **__ Introduction: __**

The Bacterial Protein – (Expression, Purification & Characterization) lab expressed, purified and characterized the bacterial pGEM-gbr22 protein. PGEM-gbr22 is a plasmid vector that was used throughout the experiments that encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef. Biological technology and biomedical science use recombinant proteins for expression in E. coli. E. coli was used as an expression host for proteins from higher organisms was used in the purification process to separate proteins for experimental work [1].The three main sections performed for this lab were protein expression, purification, and characterization which characterized the bacterial purple protein and also were able to find the concentration of the purified protein using the Nanodrop spectrophotometer and Beer’s Law. The main focus during the Protein Expression lab was to transform competent cells, grow the E.coli cultures to express the purple protein and harvest the cells by centrifugation. E. coli was transformed with the plasmid protein by interacting with the ampicillin antibiotic to form a drug resistance. The essential objective of the protein purification section was to lyse the E.coli cells that express the purple protein and clarify and purify the protein using Ni-NTA affinity chromatography.

The final purity of the protein can be optimized by controlling the ratio of recombinant protein to the column size; lower-affinity contaminants could be competed with a relative excess of the histidine-tagged recombinant protein [1]. The plasmid has six histidine residues attached at the C-terminus, which allows a fast and efficient purification of the protein [2]. Protein Characterization was used to purify the protein using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and UV-Vis spectroscopy. The plasmid vector pGEM-bgr22 protein can be secluded from the E. coli bacteria but it will not be fully secluded from some of the substances of the host bacteria in which it grew in. The entire Bacterial Protein lab contributed to the expression of certain proteins in bacteria that could help in specific drugs that could target a particular bacterium and study human proteins without requiring the use of human muscles and tissues.


 * **__ Materials & Methods: __**

The Bacterial Protein was examined in three different labs consisting of Expression, Purification and Characterization. Before each lab was conducted, safety precautions were taken into place. Gloves, goggles and a lab coat were worn at all times; bacterial waste was disposed properly and glassware was cleaned thoroughly with alconox, water and 10% bleach. During the Bacterial Protein Expression lab, three plates were harvested in a 37˚C incubator overnight. One plate was the experimental plate which contained DNA and LB ampicillin, the second plate was the control plate that did not contain any DNA and ampicillin, and the last plate was called the sneeze plate which contained a swab of saliva and LB ampicillin. The following day, a single colony was dispensed on the LB/Agar plate and expressed in a large culture in two 125 ml Erlenmeyer flasks. After the overnight incubation, the culture was centrifuged for 16-24 hours in order for the culture to grow. The next day our culture was turbid and purple in color, producing two cell pellets. The pellets weight was approximately 0.59 and 0.62 grams. 50 microliters was of the culture was dispensed into a tube called sample 1 and was stored. The two cell pellets were dispensed into a phosphate buffered saline and lysozyme was added. During the Purification process, the E. coli cells were lsyed with Benzonase to reduce the viscosity by digesting the DNA/RNA in the mixture. 50 microliters of the supernatant was placed into a tube called sample 2 and was stored. The lysate was clarified using a centrifuge and was filtered with a syringe to remove extra particles. The Ni-NTA affinity purification was used to purify the protein and made into elutions 1 and 2. During the Ni-NTA stripping procedure, 50 microliters of the flow through the waste and wash was taken and labeled as sample 3 and4. 50 microliters of elution 1 and 2 was taken and labeled as sample 5 and 6. The Nanodrop spectrophotometer was used to measure the maximum wavelength and absorbance of the purified protein (elution 1). The Beer’s Law was used to find the concentration of our protein at 280 nm and 574nm. In the Characterization lab, a 6x gel loading buffer was added to samples 1-6 that were gathered from the Expression and Purification labs. They were placed into the precast gel and then the gel was placed into the Mini-PROTEAN electrophoresis tank that contained 1x TGS buffer. After the gel electrophoresis was done, the gel was removed and stained it using the Imperial protein stain for 1 hour. The gel was washed the gel 3 times using nanopure water. The next day the vacuum was used to dry the gel. All the data collected was saved and analyzed.


 * **__ Results: __**

In the Bacterial Protein- (Expression, Purification & Characterization) labs, I was able to overexpress a recombinant protein in bacteria. I learned how to transform bacterial cells with a DNA plasmid and grow a started culture of bacteria (purple gbr22 protein). These labs enabled me to break open the bacterial cells to release the soluble proteins and also purify the protein. Gel electrophoresis, Ni-NTA affinity helped to analyze the samples and estimate the molecular weight, purity and yield of the bacterial gbr22 protein.

** Protein Expression Lab ** "Figure 1: Control plate from the Protein Expression lab that did not contain any DNA or LB Ampicillin. This plate was uncontaminated because the plate did not contain any colonies."

"Figure 2: The plasmid vector pGEM gbr22 ampicillin plate that contained the bacterial organism BL21 (DE3) after it had been grown overnight. The bacterial colonies were formed and appeared to be a purple color."

"Figure 3: Two labeled flasks that contained the purple culture consisting of LB and Ampicillin and the pGEM-gbr22 and BL21 (DE3) after being harvested overnight in the shaking incubator." "Figure 4: A 50 ml conical tube which contained cell pellet number 1, weighing 0.59 grams, that was formed after spinning the cells down in the large benchtop centrifuge for ~10 minutes centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius." "Figure 5: A 50 ml conical tube which contained cell pellet number 2, weighing 0.62 grams, that was formed after spinning the cells down in the large benchtop centrifuge for ~10 minutes centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius." ** Protein Purification Lab **

Figure 6: Elutions 1 and 2 from the Protein Purification lab that consisted of the bacterial protein after it had been purified using the combination of chromatography." "Figure 7: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 280 nm and the maximum absorbency level was at 0.520. N=2 " "Figure 8: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 280 nm and the maximum absorbency level was at 0.509. N=2 " "Figure 9: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 574 nm and the maximum absorbency level was at 0.127 N=2 " "Figure 10: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 574 nm and the maximum absorbency level was at 0.148 N=2 "  **Beer's Law was used to find the concentration of the protein at 280 nm and 574 nm.**    A= Ebc   A is absorbance   E is molar absorptivity (with units L mol-1 cm-1)   b is path length (with unit cm)c is concentration (with units mol/L)
 * Determining the concentration of the protein in mg/ml at wavelength 280nm:

Absorbance = [(0.520+ 0.509)/2] = 0.514 Extinction Coefficient = 38850 g/mol C=(0.514/38850)= 0.00001323M (0.00001323mol/L) x (1L/1000ml) x (38850g/mol) x (1000mg/1g) = 0.514 mg/ml Absorbance = [(0.148+ 0.127)/2] = 1.375 Extinction Coefficient = 118300 g/mol C=(1.375/118300)= 0.000011623M (0.000011623mol/L) x (1L/1000ml) x (118300g/mol) x (1000mg/1g) = 1.375 mg/ml
 * Determining the concentration of the protein in mg/ml at wavelength 574nm:
 * __Determining the yield Maximum wavelength__

280nm wavelength (0.514mg/ml) x 5 ml=2.5mg 574nm wavelength (1.375mg/ml) x 5ml=6.875mg

**Protein Characterization Lab** "Figure 11: The Bio-Rad precast gel after it was removed from the Mini-PROTEAN electrophoresis tank. It was stained for 1.5 hours on the orbital shaker and was destained with nanopure water."



"Figure 12: This is the Molecular Weight Marker Protein Ladder that was used to estimate the MW of the purified protein which was approximately 25 kDa."
 * **__ Discussion: __**

The Bacterial Protein – (Expression, Purification, & Characterization) lab enabled me to estimate the concentration of the purified pGEM-gbr22 purple protein at the maximal wavelength, 574 nm and at 280 nm. The estimated concentration of the protein at 280 nm approximately 0.515 mg/ml and the estimated concentration at 574 nm approximately 0.51mg/ml. Using the molecular weight standard ladder, the estimated molecular weight of the characterized protein was ~25 kDa. The estimated purity was about 12.5 because the yield was about 50% of the molecular weight. There were 4 bands present in the sample 5 lane. Lysozyme is an enzyme that was used to break down the bacterial cell walls to improve protein or nucleic acid extraction efficiency. Benzonase/Cyanase is a nuclease/enzyme that was used to reduce the viscosity by digesting the DNA/RNA in the mixture. Sample 1 was collected during the protein expression section and contained 50 ul of the protein culture after being harvested overnight. During the Protein Purification section samples 2-6 were collected. Sample 2 consisted of 50 ul of the lysate which contained the Benzonase enzyme. Sample 3 contained 50 ul of the flow through waste solution that was collected from the Ni-NTA affinity purification. Sample 4 consisted of 50 ul of the flow through wash solution that was collected from the Ni-NTA affinity purification. The Wash solution contained 10 ml of final concentration 1x PBS with 20 mM imidazole. Sample 5 contained 50 ul of Elution 1. Elution 1 consisted of 10 ml of final concentration1x PBS with 250 mM imidazole. Sample 6 contained Elution 2. The HIS tag system is when six histidine residues are attached to the C-terminus, which can be easily used to separate the protein from other cellular proteins. The histidine residues will bind to divalent cations such as nickel that can be immobilized on a column matrix such Ni-NTA agarose. The protein can be released from the Ni-NTA agarose by adding imidazole, which essentially competes with the histidine residues for metal binding. The affinity of the 6xHis-tagged protein for divalent nickel ions is the basis for the purification procedure. Some human errors that may have occurred were measuring the amounts of reagents needed. Measuring the wrong amounts of reagents could have caused problems in our culture bacteria. An error that occurred was not being able to locate our sneeze (fun) plate which delayed our lab work time. The two cell pellets had different weights which might have been caused by an irregular distribution of cells in the LB-ampicillin media. An error that occurred was an uneven distribution of certain amounts of the samples into the gel wells in the cassette for electrophoresis which became contaminated, causing the gel to have irregular colored bands. After the drying process, our gel had broken.


 * **__ Conclusion: __**

In the Bacterial Protein- (Expression, Purification & Characterization) labs, our pGEM-gbr22 protein was expressed, purified and characterized. We were able to convert proficient bacterial cells with DNA plasmid and grow a starter culture in bacteria. The lab allowed us to learn how to use an incubator for expressing overnight culture. This lab enabled the bacterial cells to be broken up to release the soluble proteins and remove the insoluble cell particles by centrifugation. The Nanodrop spectrophotometer and Beer’s Law was used to estimate the concentration of the pGEM-gbr22 protein and the Ni-NTA resin and the affinity tag was used to purify the protein. Protein characterization permitted me to use gel electrophoresis to analyze the samples from the 2 labs (Expression and Purification) and separate the proteins in each sample. Using the Molecular Weight Standard Ladder, the molecular weight of the characterized protein was approximately 25 kDa. This lab will help in VDS by searching drugs that could inhibit the purple protein that is extracted from bacteria and also to be able extract protein from other various bacterial cells in humans or animals. In addition to future experiments by collecting samples from different purification steps to be analyzed, estimate future molecular weights of other protein and help create drugs to target particular bacteria.


 * **__ References: __**

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[2]. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/protein_purification/index.html (accessed Apr 14, 2011).