Ramya+R

= Expression, Purification, and Characterization of gbr22 =

__ Introduction: __

 Recombinant proteins are essential to many areas of biological research as they allow for researchers to analyze their structure and function while growing them in an easily accessible environment. In the process of analyzing a protein, it is often necessary to first express the protein and purify it so that it can ultimately be characterized. “The objective of recombinant protein expression is usually to produce a sample that supports a certain biochemical or biological activity, such as enzyme catalysis or protein-ligand interactions” [1]. Expressing a protein involves the overexpression of a protein that is in very low abundance in its original organism, and with this increase in protein production comes the ability to easily purify the protein for further studies. When the protein has been completely expressed, it is ready to be purified. Protein purification involves lysing the cells that express the protein and clarify the protein through centrifugation. Then, the protein can be purified by using chromatography. Finally, the protein can be characterized by using the methods of gel electrophoresis to compare the staining of the protein to a specified protein ladder. In this lab, protein is grown within a bacteria, where E.coli serves as a recombinant host for the protein, pGEM-gbr22. Once the protein is cultured and retained, it is then purified using Ni-NTA chromatography, and eventually it is characterized using gel electrophoresis. Ultimately, this lab will allow us to use these methods to calculate the molecular weight, purity, and yield of the protein.

__ Materials & Methods: __

In order to carry out the expression of the protein, the main materials that are necessary are an experimental Agar plate and a control plate. The experimental plate will contain the antibiotic ampicilan as well as the DNA plasmid, BL21(DE3) pGEM-gbr22, and the control plate will only contain ampicilan. Once the plasmid is planted on the plate, both of the plates can be stored in an incubator overnight. When the protein has produced a successful culture, part of one protein colony can be placed in LB broth and stored in the shaking incubator for 16 to 24 hours so that there will be sufficient time for a starter culture to grow. When the starter culture has grown in the LB broth, forming a murky purple liquid, the protein can now be purified. Once the starter culture has been centrifuged to create a pellet of condensed cells, the lysate can be filtered and using batch and column chromatography and Ni-NTA resin. Then prepare two elution buffers by washing the pellet with imidazole, and complete the process by using the Nanodrop spectrometer to gather information about the absorbance of the first elution at a standard wavelength of 280nm and a maximum wavelength of 574 nm. Lastly, in order to complete the characterization of the protein, it is necessary to use a Mini-PROTEAN electrophoresis tank to complete the gel electrophoresis of the protein. Load a gel cassette into the tank and place each of the collected samples into the wells of the cassette. Run the gel at 200 V and then stain the gel using Imperial protein stain, and once the gel has been stained, allow it to dry in a vacuum before analyzing the staining and making comparisons between the ladder and the protein.

__ Results: __



















__ Discussion: __

Using Beer’s Law, A=Ebc, the molecular weight and yield of the protein can be calculated. The literature value of the molecular weight of gbr22 is 25 kDa.  extinction coefficient = 38850 M-1 cm-1  Molecular weight = 25794.2 g/mol.

 A = 0.5035 at 280 nm, E = 38850, b = 1 cm A = Ebc 0.5035 = (38850)(1)c  c = (1.296E-5)(25794.2)  c = 0.334 g/mol.  yield = cV yield = (0.334)(4.75 ml)  yield = 1.5865 mg

 A = 0.0945 at 574 nm, E = 118300, b = 1 mm  A = Ebc 0.0945 = (118300)(1)c  c = (7.988E-7)(25794.2) <span style="font-family: 'Arial','sans-serif';"> c = 0.021 g/mol. <span style="font-family: 'Arial','sans-serif';"> yield = cV <span style="font-family: 'Arial','sans-serif';"> yield = (0.021 g/mol)*(4.75 ml) <span style="font-family: 'Arial','sans-serif';"> yield = .0979 mg

The lysozyme used in this lab helped break down the cell walls of the bacteria so that the proteins could grow, and benzonanse was used to remove DNA from the starter culture of the protein. The samples taken throughout the lab represent different steps in protein expression and purification. Sample 1 came from the starter culture made during protein expression. When the cell was first lysed in the beginning of protein purification, sample 2 was obtained. Samples 3 through 6 were collected as the cell pellet was being washed and filtered with the imidazole and elution buffer. Comparing the calculations at the two different wavelength, the protein has a much smaller concentration, 0.021 g/mol, at 574 nm than it did at 280 nm, 0.334 g/mol. Based on the dry gel image, the molecular weight falls between 25 kDA and 35 kDa, so I would estimate the molecular weight of this protein to be about 30 kDa.

__ Conclusions: __

The purpose of this lab was to overexpress a protein so that it could be further studied and ultimately be characterized by its molecular weight and yield. Any sources of error in this lab most likely came from the improper handling of a step. The expression, purification, and characterization of a protein involves many meticulous steps that, if one goes incorrectly, the entire process can easily be damaged or contaminated. In the future, the skills obtained through this multistep lab will help with the extensive studies of proteins.

__ References: __

1. Protein production and purification. Nat Methods. 2008 February ; 5(2): 135–146. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178102/pdf/nihms194747.pdf.