Expression of Recombinant Green Fluorescent Protein (rGFP)

Expression and Purification of recombinant Green Fluorescent Protein (rGFP) from E. coli using Ni²⁺-Agarose Column Chromatography.

• Andrea Bustamante
• Janakikeerthika Darmarpandi

Abstract

Green Fluorescent Proteins are vital components of bioluminescence in marine animals. There unique ability to withstand and recover from harsh conditions and regain fluorescence was of great interest. The purpose of the following set of experiments was to express and purify a His₆-Xpress epitope tagged recombinant form of Green Fluorescent Protein grown and harvested from E. coli. The desired protein is initially released into solution using the properties of freeze-quick thaw cycles that then help release the contents of the nucleus of neighboring bacteria following a chain reaction. It is then submitted through a Ni²⁺-agarose affinity chromatography column where the target protein was purified. The resulting wash and elution fractions where run through a Bradford assay, SDS-PAGE/Coomassie blue staining, and a Western blot to determine the molecular weight of the protein to be 32kDa. The overall specific activity was determined to be 433000 RFU/ mg of total protein with a resulting 20 percent purity. The results show that expression and purification of rGFP from bacterial cells was possible.

Introduction

Aequorea victoria is a jellyfish capable of producing a green fluorescent light when Ca²⁺ ions activate a photoprotein, known as aequorin, which excites Green Fluorescent Protein (GFP). Wild type GFP is a 27kDa, homodimer composed of 238 amino acid residues that absorbs light at an excitation wavelength of 395nm (blue light) and emits light at an emission wavelength of 510nm (green light). Aequorea victoria GFP has a distinctive three dimensional structure that encases a chromophore (formed by cyclization of Ser65-dehydrogenized Tyr-Gly67) and allows for stability under harsh conditions (Prasher, 229-230.)

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. This structure allows for regaining of fluorescence even after the protein has been denatured upon removal of the denaturant. Therefore, GFP’s are extremely stable to changes in pH, temperature, oxidation and reduction, and chemical reagents (Pan, Pickett, and Rippel 225.)

Poly-histidine tags involve addition of a series of histidine residues to the N or C terminus of a protein of interest. Poly-histidine tags are affinity tags that serve to facilitate protein purification by exploiting the positively charged histidine residue’s affinity for negatively charged columns. This series of experiments involved a six repeat histidine codon contained within a DNA plasmid which resulted in a recombinant Green Fluorescent Protein that contained a six residue histidine tag located at the N-terminus. The His­₆ tagged recombinant Green Fluorescent Protein was then subjected to Ni²⁺-agarose column affinity chromatography.

Ni²⁺-agarose affinity chromatography allows for the purification of poly-histidine tagged proteins due to the selectivity and affinity of the Ni²⁺-agarose matrix for His₆ tagged proteins. rGFP binds the column due to the interactions between the His₆ tagged proteins in the mobile phase with the metal Ni²⁺ ions immobilized within the matrix in the stationary phase. The Ni²⁺ ions contained within the matrix are capable of binding electron rich molecules including histidine residues and allowing most other molecules to pass unbound. This results in the binding of the desired protein to the column and the purging of most undesired proteins and contaminants from the column into wash fractions (Ninfa, et al. 100-101.) The column was then subjected to imidazole, which competes with rGFP for Ni²⁺ ion attachment, and this allows for the elution of the target protein.

Due to its unique properties, isolation of GFP was of great interest and expression and purification were the main focus of the following series of experiments. A suitable way to accomplish this was devised using the combination of poly-histidine tagging and affinity chromatography. The purpose of this experiment was to express and purify a six-Histidine tagged recombinant form of Green Fluorescent Protein from E. coli through the use of Ni²⁺-agarose affinity chromatography. After expression and purification, a Bradford assay was performed to estimate total protein amount. This was followed by SDS-PAGE/Coomassie blue staining to determine purity and molecular weight. The confirmation of the presence of rGFP was done using the Western Blot.

Materials and Methods

Growth of G strain

In a test tube, 10ml of liquid LB growth media containing 100ug/ml Amp and 25ug/ml Cam was inoculated with a single bacterial colony of strain G (BL21(DE3)uv>) and was allowed to grow overnight at 37°C. The culture was shaken until saturated. In a flask, 500ml of liquid LB media (pre-warmed to 30°C) was inoculated with about 4 ml of the saturated overnight culture (or until the 500ml culture reached an OD₆₀₀ reading of 0.1) and allowed to grow at 37°C until the OD₆₀₀ reading reached 0.5. At approximately OD₆₀₀ ~0.5, or time zero, 1ml of the culture was harvested into a 1.5ml centrifuge tube and pelleted. The supernatant was discarded and the “G0” pellet stored at -20°C for later use. The remaining culture was induced with 1mM IPTG and allowed to grow. After 3 hours, 1ml of the culture was harvested into a 1.5ml centrifuge tube and pelleted. The supernatant was discarded and the “G3” pellet stored at -20°C for later use. An additional 15ml of the IPTG induced culture was harvested into a 15ml centrifuge tube and pelleted. The supernatant was discarded and the “G3-15ml” was stored at -20°C.

Preparation of rGFP Crude Extract

Immediately after removal of the “G3-15ml” pellet from freezer, breaking buffer [10mM Tris, pH 8.0; 150mM NaCl] was added into the centrifuge tube. The breaking buffer was pipetted up and down (being careful not to introduce air) until pellet had thawed and homogeneity was reached. The solution was transferred into a 1.5ml centrifuge tube, vortexed for 5 minutes, labeled and placed in 37°C water bath for 10minutes after which the centrifuge tube was transferred to a rotating platform shaker in a dry air 37°C incubator for 20 minutes. After lysis, the mixture was centrifuged at 14000xg, 4°C, for 10 minutes. In a dark room in the presence of a hand held UV light, the fluorescence of the pellet and supernatant where observed the recorded. The supernatant was then decanted and care was taken not to get the pellet back into the supernatant as centrifugation would be required if this did occur. This supernatant was the GCE (rGFP crude extract)

Preparation of Ni²⁺-agarose Column

In a 3ml plastic syringe, enough glass wool was placed into the well to cover up to the 1/4 ml marking. The syringe was secured onto a ring stand and placed perpendicular to the ground. About 100ul of breaking buffer was pipetted into the top of a closed luer-lock and allowed to overflow. 1ml of buffer was then pipetted into the syringe column and the luer-lock was immediately screwed onto the syringe. An additional 2ml of breaking buffer was added to the column and several drops of buffer were allowed to flow out. The luer-lock was then returned to the closed position. A total of 500ul of breaking buffer was added to the column and then 1ml of a 0.5ml bed volume Ni²⁺-agarose slurry was added to the column. The luer-lock was opened and agarose matrix was allowed to “gravity pack.” The column was pre-equilibrated with 5ml of breaking buffer and then the luer-lock was returned to the closed position.

Ni²⁺-NTA Chromatography Separation Procedures

100ul of GCE was transferred into a centrifuge tube, labeled, and set aside.

Breaking buffer was added to remaining GCE if content was less than 1ml. GCE was slowly applied to the Ni²⁺-agarose column and allowed about 5-10 minutes for protein to bind to column. The luer-lock was opened and 0.5ml of effluent was collected into 1.5ml centrifuge tube and labeled W1. This was repeated with the subsequent effluent labeled W2.The column was then observed under an ultraviolet light and fluorescence recorded. The column was then washed with 4ml of buffer in 0.5ml increments. The effluent was collected and labeled W3 to W10. The column was then washed again with a total of 5ml of breaking buffer. This effluent was discarded. A total of 5ml of elution buffer containing 10mM Tris, pH 8.0; 150mM NaCl, 300mM imidazole was added to the column in 0.5ml increments. The eluents were collected and labeled E1-E10.The column was then observed under a UV light and the fluorescence recorded. The W1-W6 and E1-E6 fractions were also observed under UV light and their fluorescence recorded qualitatively.

Determining Total Protein Amount

A standard curve was created using six different samples of Bovine Serum Albumin (1mg/ml) of known amount. The amounts of BSA used all had a final volume of 50ul and included 0ug, 3ug, 5ug, 10ug, and 20ug total proteins. A total of 1ml of Bradford reagent was added to each, vortexed, and allowed to incubate for 10 minutes. The results where read using 200ul in a microtiter dish and read using a microplate reader set to 595nm. The results where plotted on a graph as absorbance (595nm) vs. BSA (ug) and a best fit line was drawn. The Bradford assay was then performed once on the W1-W6 and E1-E6 samples. Any samples whose absorbance fell outside the standard curve were repeated less sample in the assay. Once all samples fell within the standard curve, the Bradford assay was repeated two more times for each sample. The total protein amount was then extrapolated from the standard curve using the absorbance values.

Estimating Purity and Molecular Weight

The SDS-PAGE was prepared using a 12 percent resolving gel that was poured between the Bio-Rad glass plate “sandwich” and allowed to polymerize. A 5 percent stacking gel was prepared and added on top of the resolving gel, a comb was inserted, and the gel was allowed to polymerize. Once that polymerized, the combs were removed and the electrophoresis tank was set up. 15ul of G0, G3, GCE, W3, W4, E2, and E3 samples were added to the SDS-PAGE along with a standard molecular weight ladder. The samples were electrophoresed at 200volts for 45 minutes. The gel was then stained using Coomassie blue dye and the stain removed.

Confirmation of rGFP

2-β-mercaptoethanol was added to the centrifuge tubes containing the G0, G3, GCE, W3, W4, E2, and E3 samples and were loaded along with a molecular weight ladder and electrophoresed as described above. The stacker was removed and the resulting gel set up for transfer onto a nitrocellulose membrane for Western Blot analysis. The overall setup required a “building up” of components with the positive electrode base on the bottom, followed by filter paper soaked in transfer buffer, nitrocellulose paper above that, the SDS/PAGE layer, another layer of filter paper soaked in transfer buffer, Western blot solution was poured over all the components, and finally the negative electrode lid was locked into position. To ensure transfer, the nitrocellulose gel was stained using Ponceau S and allowed to incubate for two minutes on a rocker and then destained using ddH₂O. The membrane was then blocked using 5% non-fat dry milk/TBS solution and incubated for 30 minutes on a rocking platform. This was then and washed three times with 0.05%Tween 20/TBS with 5 minutes of incubation between each wash. It was then probed with mouse IgG anti-Xpress epitope MAb solution and allowed to incubate for 45 minutes. The 0.05%Tween 20/TBS wash was repeated in triplicate. A secondary probe using sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution was performed as above and then washed in triplicate. The nitrocellulose gel was developed using TMB until desired intensity was reached and development was stopped with water and results recorded immediately.

Results

The expression of the target protein was doubly repressed in the G0 (uninduced) sample of E. coli. First, the Lac repressor protein binds to the lac operator and prevents transcription by T7 RNA polymerase (Garrett and Grisham 915-916). Second, T7 RNA was repressed by lysozyme protein that binds to T7 RNA polymerase and inhibits transcription. Expression of rGFP in the G3 (3 hour post induction) sample was made possible through the use of IPTG (Garrett and Grisham 914.) The purpose of IPTG was to repress the Lac repressor which resulted in T7 RNA polymerase being able to transcribe DNA downstream of the T7 promoter and expression of His₆-Xpress-GFP_uv, resulting in the fluorescent capable recombinant Green Fluorescent Protein. (Figure 1)

This resulting recombinant GFP is a 279 amino acid protein. rGFP has a six Histidine tag at its N terminus between amino acids 5 and 10, an Xpress epitope between amino acids 24 and 31, Green Fluorescent Protein between amino acids 39 and 277, and a 3 amino acid end tag between amino acids 277 and 279. The chromophore is found between amino acids 103 and 105 in the DNA sequence. (Figure 2)

Results of Ni²⁺-agarose affinity chromatography and Bradford assay indicated that the E3 (elution 3) sample contained the most rGFP activity with approximately 18,600 RFU (relative fluorescent units) and an estimate 43ug of total protein. The specific activity calculated for the sample was 433000 RFU/ mg of total protein. (Figure 3)

The SDS-PAGE/Coomassie staining gave an estimate molecular weight for rGFP of 32kDa based on a total traveled distance of 2.3cm along the SDS/PAGE. The overall purity of the band was approximately 20 percent. The higher molecular weight band was most likely contaminants at about 45kDa and the lower molecular weight band was possibly a result of the degradation of the c-terminus at 27kDa. (Figure 4)

Western Blot indicated prominent bands in the E3, E2, GCE, and G3 lanes. Lanes W4 and W3 showed very light bands and lane G0 shows an absence of bands. All visible bands appear at about 32 kDa and therefore confirm the presence of rGFP. (Figure 5)

Conclusion

The successful expression and purification of recombinant Green Fluorescent Protein is significant in the scientific community due to the possible uses for it in the future. Green Fluorescent Protein is significant because it provides an inexpensive and relatively easy method of detection. The possibility for real time detection means result could be obtained in real time. Future experiments will focus on linking rGFP to proteins during transcription and translation. This would result in a desired protein with a GFP tag whose fluorescence can then be used for identification. This should result in the ability to locate a target protein using the fluorescence of rGFP.

Future applications of GFP could include incorporation into the genetic code of small mammals. These could encode fluorescent neurons which in turn could help further research in areas such as nerve tissue regeneration or other advances in neurobiology. Its unique properties of endurance could be exploited to understand how it can endure harsh environments and still regain functionality after remediation. This would have significant applications in molecular and cellular biology in understanding cellular degeneration and how help patients with diseases involving cellular degeneration.

Bibliography

Pan, Jing, Elizabeth Pickett, and Scott Rippel. Biochemistry Laboratory Lecture Notes. Dallas: UTD copy center, 2013. 225-289. Print.

Pan, Jing, Elizabeth Pickett, and Scott Rippel. Biochemistry Laboratory Manual. Dallas: UTD copy center, 2013. 38-77. Print.

Prasher, Douglas C., Virginia K. Eckenrode, et al. “Primary Structure of the Aequorea victoria green-fluorescent protein.” Gene. 111. (1992): 229-233. Print.

Garrett, R., and Charles M. Grisham. Biochemistry. 4th ed. Belmont, CA: Brooks/Cole, Cengage Learning, 2010. Print.

Ninfa, Alexander J., and David P. Ballou. Fundamental laboratory approaches for biochemistry and biotechnology. Bethesda, Md.: Fitzgerald Science Press, 1998. 89-107. Print.