JeanetteC

= pGEM-gbr22 in // E. coli // to Express, Purify, and Characterize the Protein =

__Introduction__
Recombinant proteins are used throughout biological and biomedical science. These proteins are generally suitable for expression in // E. coli //. // E. coli //is used as an expression host for proteins from higher organisms, which increases the yield of soluble protein. It is cost-effective and convenient by allowing a fairly comprehensive analysis within a relatively short period of time. For high-level protein production purposes, BL21(DE3) is an appropriate // E. coli //strain. The three main steps performed for this lab were protein expression, purification, and characterization. Before protein expression is done, the gene is cloned and inserted into an expression plasmid. The presence of the gene is confirmed and the gene is sequenced to ensure the cloning process did not introduce mutations. The plasmid used in this experiment, pGEM-gbr22, encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef. The plasmid has a gene for ampicillin resistance. // E. coli //BL21(DE3) was transformed with the plasmid, and grown with antibiotic to select for colonies with the gene for antibiotic resistance. Small-scale test expression is used to determine which of the clones actually produces soluble protein, to establish the optimal scale for the large-scale growth. After the bacterial cells are grown, they are harvested The next step is purification. The plasmid has six histidine residues attached at the C-terminus, which allows a fast and efficient purification of the protein. Immobilized metal ion affinity chromatography (IMAC) has a strong, specific binding to amino acids, particularly histidine, to metals. IMAC also has mild elution conditions and the ability to control selectivity with low concentrations of imidazole in chromatography buffers. The histidine residues of gbr22 will bind to divalent cations, such as nickel. Ni-NTA (nitriloacetate agarose) will be used to bind the protein, and then released by adding imidazole. The last major step is characterization, which reduces the risk of wasting resources of protein material of inadequate quality and ensure that different batches of the same protein have similar properties. Characterization determines whether the right protein was purified, contamination, and estimate of approximate protein concentration. The most common technique used to follow expression and purification steps is SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The protein samples are at one end of a porous gel and an electrical field is applied across the gel using a power supply. It denatures proteins and passes a negative charge that is proportional to its mass. The distance migrated by the protein through the gel is related only to its mass. This allows an estimation of its mass to be made by comparing it to molecular weight standards. When the gel is stained, the intensity of the bands will usually be proportional to the amount of protein.

__Materials and Methods__
Bacterial protein expression First day the competent bacterial cells were transformed. Three LB Agar plates were prepared and incubated; an experimental plate with DNA, a control plate with no DNA, and a ‘fun’ plate that was coughed on. BL21(DE3) competent bacterial cells from NEB (New England Biolabs, Ipswich, MA) was added to the experimental and control. The plasmid, pGEM-gbr22, was added only to the experimental, and SOC media was added to the experimental and control plate. Second day, a starter culture was grown in LB supplemented with ampicillin (AMP). Third day, by adding AMP to the LB and incubating made protein expression in a large culture. Fourth day, sample 1 was collected, and cells were harvested. A PBS working solution was made and added to the cell pellet, an even suspension of cells was obtained, and lysozyme was added. Protein purification // E. coli // cells were lysed, Benzonase was added, the lysate was clarified by centrifuging, and sample 2 was collected. The liquid supernatant was collected, leaving the cell debris pellets behind, and was syringe filtered. Wash and elution buffers were prepared. Ni-NTA affinity purification was done using a combination of batch and column chromatography. The protein was bound to the resin with Ni-NTSA resin/buffer mix, waste was collected, followed by gathering sample 3. Proteins loosely bound to the resin were removed by washing with imidazole in PBS. Wash was collected, and a sample 4 was taken from the wash. Gbr22 protein was released from the Ni-NTA resin by using a buffer with a high concentration of imidazole; elution 1 was collected. Elution buffer was added; elution 2 was collected. Sample 5 was collected from elution 1, and sample 6 was collected from elution 2. Using Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to estimate the concentration of final purified protein. Protein characterization First day, SDS-PAGE gel samples were prepared. Samples 1-6 were added gel loading buffer. The electrophoresis module was assembled and filled with TGS buffer. PageRuler MW standard was loaded into the second well, and each protein sample was loaded into each of the remaining wells. The gel electrophoresis was performed, and the gel was removed and stained. Second day, the gel was placed on Whatman paper and covered with cellophane, and dried.

__Results__
280nm: A=Ebc c=A/Eb c=.238/(1cm)(38,850M-1cm-1)= 6.126x10-6 M 6.126x10-6mol/L x 25,794.2g/mol x 1L/1000ml x 1000mg/1g= 0.158 mg/ml Elution 1: 0.158 mg/ml x 5ml= 0.79mg Elution 2: 0.158 mg/ml x 4.5ml= 0.71mg Total yield: 1.5 mg 574nm: A=Ebc c=A/Eb c=.4/(1cm)(118,300M-1cm-1)= 3.38x10-6 M 3.38 x10-6mol/L x 25,794.2g/mol x 1L/1000ml x 1000mg/1g= 0.087 mg/ml Elution 1: 0.087 mg/ml x 5ml= 0.44mg Elution 2: 0.087 mg/ml x 4.5ml= 0.39mg Total yield: 0.83 mg

__Discussion__
The purple colonies of // E. coli // that grew in the experimental LB Agar plate with AMP and DNA, were resistant to ampicillin. The molecular weight of gbr22 is 25,794.2 g/mol. The absorbance of gbr22 protein at 280nm was 0.37abs, and the extinction coefficient is 38,850 M-1cm-1. The wavelength in the visible region where gbr22 protein absorbs maximally is at 574nm with 0.4abs and the extinction coefficient is 118,300. Using the absorbance measured at 280nm, 0.37abs, the extinction coefficient, 38,850 M-1cm-1, and Beer’s law was used to determine the concentration of gbr22, 0.158 mg/ml. The concentration of gbr22 at 280nm and the volumes of purified protein collected were used to calculate the yield of purified protein, which was 1.5mg. Using the absorbance at 574nm, 0.4abs, the extinction coefficient, 118,300, and Beer’s law was used to determine the concentration of gbr22, 0.087 mg/ml. The concentration of gbr22 at 574nm and the volumes of purified proteins collected were used to calculate the yield of purified protein, which was 0.83mg. The concentration of gbr22 at 574nm does not correlate with its concentration at 280nm. Tryptophan of gbr22 could have been in high amount and absorbed more at the maximal wavelength. The gel electrophoresis and staining indicates that protein samples with a greater molecular weight have a higher band than samples with a lower molecular weight. The estimated MW of gbr22 was 25 kDa (kilodaltons) using the standards as a reference. Four protein bands were present in the sample 5 lane. The estimated purity of the final protein, sample 5, was about 25%. Since the protein had more than one band, the protein was not pure, indicating contamination.

__Conclusion__
The plasmid, pGEM-gbr22, was inserted into // E. coli // BL21, the host bacteria to overexpress the recombinant protein. The protein gbr22 was purified and samples were collected to examine using gel electrophoresis. Using electrophoresis and spectroscopy results, the molecular weight of the protein, gbr22, was estimated. The purity and yield of the final purified protein product was calculated and the protein solution concentration was estimated. Protein expression, purification, and characterization apply to virtual drug screening, since drugs are constantly researched and developed to inhibit proteins or increase protein synthesis, such as the production of human insulin to treat diabetes. Once the information on the protein is collected, drugs can be screened to inhibit the protein, and lead to a narrowed down list of drugs. These drugs can then be tested in the lab to see if they actually work.

__References__

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