Aakash Sur

Expression and Purification of Montipora efflorescens gbr22 in E. coli

Introduction: Studies of the proteome provide the most fundamental understanding of the molecular mechanisms of life. Experiments of protein functionality, targeting, and structure, define these mechanisms, but require the in vitro production of proteins and enzymes. To achieve this, recombinant technology has evolved to produce target proteins in various vector organisms. Generally, the recombinant expression process begins with construction of cDNA from genomic DNA encoding for a specific gene product. This construct can be incorporated into the genome either by bacterial transformation of a plasmid or bacteriophage transfection. Here, we utilize a competent Escherichia coli strain that has been treated with calcium ions to allow for the uptake of foreign plasmid DNA.1 The plasmid contains several genes, the cDNA fused to an identifiably tag such as an oligohistadine or glutathione Stransferase, an origin of replication, an ampicillin resistance gene, and an overexpression gene. Once the E. Coli have been transformed, they will overexpress the cDNA gene product, which can be harvested by lysing the cells and purifying intracellular contents. A common method of purification is using immobilized metal affinity chromatography (IMAC) that functions by forming coordination compounds with the functional groups of amino acids. 2 In our particular experiment, we tagged Montipora efflorescens gbr22 florescent coral protein with an Nterminal hexahistadine sequence that allows a Nickel-Nitriloacetic Acid Column to selectively bind to the expressed protein. We hypothesize that transformed E. Coli BL21 (DE3) cells will overexpress gbr22 fused his-tag, which can be used to purified the protein on a Ni-NTA IMAC
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column. Expression and Purification of Recombinant Protein: Montipora efflorescens gbr22 florescent coral protein was overexpressed in Escherichia coli BL21 (DE3) using a pGEM plasmid encoding for a hexa-histadine tag fusion construct. The transformed bacteria contained the ampR gene allowing for the selective growth of these cells in ampicillin enriched media. The bacterial colony was grown in Millers Lysogeny Broth, a concoction of 10g/L Tryphone, 5g/L Yeast extract, 0.17M NaCl, and 100 g/ml Ampicillin, at 37C. The cells were pelleted and subsequently re-suspended in a lysis buffer; 1xPBS, and 1mg/mL Lysozyme. To harvest the recombinant protein, the cells underwent a freeze-thaw cycle, and 50 units of Benzonase were added to digest DNA and RNA. The sample was then centrifuged at 14,000g for 20 minutes to pellet the cytoplasmic contents. The supernatant was extracted and filtered through a 0.22 m syringe filter and then allowed to interact and bind to Ni-NTA His-tag affinity resin. A Bio-Rad chromatography column was prepared with the Ni-NTA protein resin and washed with 20mM Imidazole and 1xPBS. Characterization of Recombinant Protein: To determine both the purity and integrity of the recombinant protein, the sample was analyzed using gel electrophoresis, using a an SDS PAGE 4%-20% gradient gel (BioRad) 10cm x 10cm. The gel was washed then stained with Imperial protein stain then allowed to destain overnight. To preserve the gel, it was placed on Whatman filter paper and wrapped in cellophane and finally allowed to dry out in a drying bed. Results:

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Figure 1: Colonies of E. coli growing. On the right is the control plate with nontransformed cells and on the left is the purple transformed colonies. Surprisingly, bacteria on the control plate which did not have the ampR gene through the plasmid still grew on the ampicillin enriched media.

Figure 2: Bacterial grown from a transformed colony.

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Figure3: The centrifuged and pelleted bacterial cells containing the overexpressed gbr22 protein which is bright purple.

Figure 4: The initial spectrographic analysis of the purified protein sample at 280nm.

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Figure 5: A duplicate spectrograph of the protein at 280nm.

Figure 6: A spectrograph of the purified protein at the maximal 574nm wavelength.

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Figure 7: A duplicate spectrograph of the purified protein at the maximal 574nm wavelength.

Figure 8: Characterization of purified fractions of gbr22 via gel electrophoresis. On the left is an illustration of the various molecular weight the protein ladder corresponds to. On the
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right is the stained gel: From left to right, the first lane is the PageRuler molecular weight standard. The second is the total bacterial lysate of the transformed cells. The third lane is the intracellular protein contents after nuclease digestion. The fourth is the waste flow-through fraction from the chromatography column. The fifth lane is the wash fraction, containing weakly bound impurities that came the Ni-NTA at 20mM Imidazole concentrations. The sixth is the first elution containing all proteins coming off the column with 250mM Imidazole. The seventh lane is the second elution fraction. Discussion: Characterizing the expressed and purified gbr22 reveals several impurities through gel electrophoresis. The intracellular contents of the bacteria can be seen in lane 1, where the total lysate is present, and in lane 2, where the protein content is present. These lanes reveal a gross overexpression of gbr22 at approximately the 25kDa band. Despite the protein purification process, there were three distinct band present in the elution; the 25kDa band being the gbr22, and the 15kDa and 10kDa being impurities. In addition, there was a relatively high background smear of proteins ranging from 100kDa to 10kDa. Given these results, we conclude that the protein sample is about 35% pure, and is not yet pure enough to conduct in vitro studies. Copurification of E. Coli protein with histidine-tagged protein is commonly reported due to contaminant proteins containing multiple histidine residues, and molecular chaperones that may bind to the resin or recombinant protein. One method of repurification is to cleave the hexahistidine site using thrombin, and then using the Ni-NTA column to reverse the chromatography process. Questions:

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1. Why do we use lysozyme? Why do we use Benzonase/Cyanase? Lysozymes are enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-betalinkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. This allows for the breakdown of the cell wall and the lysis of E. Coli to expose intracellular contents where the protein construct is present. Benzonase is a genetically engineered endonuclease from Serratia marcescen, and promiscuously digests DNA and RNA to reduce the viscosity of lysed intracellular contents to facilitate recombinant protein purification. In this case, the complete digestion of DNA and RNA allows eases gravity driven column chromatography, and eliminates their presence in gel characterizations. 2. What is different about the Wash vs. the Elution buffer? The wash buffer contains a 20mM concentration of Imidazole whereas the elution buffer contains a 250mM concentration. The lower concentration of Imidazole in the wash buffer removes proteins that are weakly associated with the Ni-NTA. Once these impurities have been removed, the highly concentrated elution buffer releases the olgiohistidine tagged proteins separately. 3. Briefly, explain how the HIS tag system works The cDNA plasmid insert for gbr22 was fused to a sequence encoding for an N-terminal hexahistidine tag. The expressed protein contained this oligohistidine which allows for
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immobilized metal affinity chromatography purification using an Ni-NTA resin. The Nitrilotriacetic acid is a chelating agent that holds the Ni2+ through four coordination bonds, leaving two open sites to bind to the imidazole ring side chain of histidine. The Ni-NTA resin in the chromatography column captures the 6xHis-tagged gbr22 and can be selectively eluted at high concetrations of Imidazole, which replace the coordination bonds of the protein. Conclusion: We expressed Montipora efflorescens gbr22 florescent coral protein in Escherichia coli BL21 (DE3) and purified it using an immobilized metal ion affinity Ni-NTA chromatography column. Characterization of the eluted purified protein solution through gel electrophoresis revealed that the competent E. coli were successfully transformed. However, it also revealed several significant impurities in solution, indicating that our purification protocol was not entirely effective. We hope to optimize the protocol in the future to ensure complete purification of gbr22. Recombinant protein expression is crucial for in vitro enzyme function assays to test the effectiveness of inhibitory compounds selected by virtual screening. References:
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