Tom+S

__ Bacterial Protein E.P.C (Expression, Purification and Characterization) __

__ Introduction: __

Recombinant proteins and DNA are becoming increasingly popular. Numerous researches are participating in finding out the new techniques or manipulating already existing DNA to generate a desired protein [1]. Usually, the gene is amplified through PCR or other gel techniques. In this particular lab, E.Coli bacteria protein would be overexpressed and purified for crystallography, enzyme inhibition assays and other lab techniques. Calculating concentration process would be accompanied with spectrophotometer and Beer's law. The objective of this lab was to overexpress, purify, and characterize the recombinant pGEM-gbr22 protein.

__ Materials & Methods: __ Safety issues were wearing gloves and a coat at all times. Materials were p-Gbr 22 protein from NEB, tubes, beakers, and other lab equipments, such as gel electrophoresis device and spectrophotometer (Thermo Scientific, Wilmington, DE). Procedure was divided into three parts: expression, purification, and characterization. First, the bacterial protein was prepared to grow. 25ul of bacteria was added into 2 transformation tubes and was added to 200ul of SOC media after a few ice and heat shock process. The bacterial was left to grow under 37 ̊C incubator. The next day, bacterial colony was added to LB broth and left at the incubator for 7 hours. Then, appropriate amount of ampicillin was added to the culture in Erlenmeyer flask and left to grow for 24 hours in the incubator. Sample 1 was gained from this culture after 24 hours. The culture was centrifuged for 5 minutes at 5000 rpm at 4 ̊C. The pellet was retrieved and dispensed. Lysozyme was added to make the final concentration of 1mg/ml. The pellet after centrifuge was weighted, which was 0.18g. Then 2 ul of benzonase was added to the solution. The solution was then centrifuged to produce sample 2. The liquid collected was syringe filtered. Meanwhile, elution buffer 1 and 2 were made (1x PBS and 20mMimidazole and 1x PBS and 250mM imidazole). The protein was then purified through column chromatography. 0.5mL of Ni-NTA resin/buffer was added to the column. The sample 3 was collected after this, and sample 4 was collected after 5 ml of wash buffer was added. Sample 5 was collected after 5ml of elution buffer and the remaining elution buffer was added and collected to be sample 6. Nanodrop spectrophotometer was used to measure the absorbance of the elution 1 sample at 280nm and 574nm. The samples (1-6) were loaded on SDS-PAGE gel for gel electrophoresis. The MW standard was used as PagerRuler by Fermentas. The gel electrophoresis process was done for 25 minutes at 200V. Then, the stained protein was placed in orbital shaker for 1.5 hours. The gel was retrieved and dried at 75 ̊C for 1.5 hours.

__ Results: __

Figure 1. The fun plate for the bacterial colony Figure 2. The bacterial colony with the DNA included. Figure 3. The bacterial colony with nothing. It was used as control.

Figure 4. The bacterial colony with purple culture. It was purple because the bacterial protein itself was purple in color.

Figure 5. Harvested cell in a form of purple pallet. The weight was 0.18g.

Figure 4. Solution extracted after addition of elution 1 and elution 2

Protein 280 trial #1 Protein 280 trial #2

Protein 574 trial #1 Protein 574 trial #2

Molecular weight of gbr22= 25794.2 g/mol The average of protein at 280nm = 0.339 absorbance A=ebc, C=A/eb= 0.339/118300= 0.0779M. The average of protein at 574nm= 0.325 absorbance -> C= 0.216M. The yield for 280nm was 0.0739x5= 0.3695 mg/ml. The yield for the maximal wavelength (574nm) was 1.08mg/ml.

Figure 5. Protein gel image before drying.

Figure 6. Cracked gel after drying process. Fermentas Pageruler Molecular Weight Standard __ Discussion: __ The lysozyme was added to break down the cell wall of the E. Coli bacteria. After breakdown of the cell wall, pGEM-gbr22 could be separated and we could further purify the only gbr22 protein. Benzonase was used to increase fluidity of the solution. Since benzonase breaks down DNA or RNA, the viscosity of the solution decreases. Sample 1 contains cell lysate. Sample 2 contains bacterial DNA after centrifuge process, and sample 3 contained after “flow through” of the Ni-NTA solution. Sample 4 contained protein and other residues after “wash buffer”. Sample 5 contained protein and other residues after addition of elution buffer 1. Sample 6 contained almost purely protein after addition of elution buffer 2. Wash buffer was made up of small amount of imidazole, while elution buffer was consisted of PBX and imidazole solution. HIS-TAG system is often used for protein purification of E. Coli. Bacterial cells are harvested via centrifugation and the resulting cell pellet is lysed. The Ni-NTA solution is used for the concept of micromolar affinity of the resin and the bacterial cell. Then, the protein samples from the bacterial cell are further purified through imidazole or elution buffer solution [2]. Overall, the protein was purified very well. The gel image was similar to the rest of the classmates. Numerous errors occurred. First, decanting the liquid part usually resulted in loss of the pellet. Also, too much buffer or imidazole solution might have been added to the solution. During drying process of the gel, the cell paper was not strong enough so that when the heater was opened after heating, the gel cracked.

__ Conclusions: __ In this experiment, pGEM-gbr22 protein was expressed, purified, and characterized. Protein samples were each isolated to compare at the end to find out how purified the protein was. The molecular weight of the purified protein was estimated to be around 27kDa. The percent of purity was concluded to be around 75-80%. The protein was left for future experiment on protein isolation or other projects. The next step might be performing enzyme assays to explore the function of protein and find out the inhibitory behavior of the protein.

__ References: __

1. SQ, Wang; HY, Zhou; Q, Gao; Quantitative detection and species identification of human Plasmodium spp. by using SYBR green I based real-time PCR. //Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi **2011,**// (6) 677-81

2. LL, Larcia; LM, Dalmacio; Bacterial diversity in Philippine fermented mustard as revelaed by 16s rRNA gene analysis. //Benef Microbes.// **2011,** (4) 263-71