Protein Synthesis Lab Report

Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.

Having removed the detergent, the protein will refold. As shown by the Anfinsen experiment the polypeptide sequence determines the folding and therefore the three dimensional structure. As the polypeptide sequence is unaltered refolding can occur through the process of nucleation aggregation and compaction. In order to test that the protein was no longer denatured, the absorbency of the solution at 412nm could be measured and compared with the graph in figure 1 above, it should match the plot of standard ovalbumin in the absence of SDS.

Since the side chains are bonded to ions in solution, they are unavailable to bond with each other. This lack of bonding amongst the side chains effects the tertiary structures of the protein, changing its shape. The tertiary structure is important because for an enzyme to work, it must have a very specific shape to fit, lock and key style, onto the substrate. As the substrate and enzyme bind the shape of the substrate molecule slightly bends. This strains the bonds of the substrate, allowing them to be broken easy.

The proteins are also added to a Laemmli sample buffer in order to give each protein a negative charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance in which the proteins travel through the gel. If it is run for too long, the proteins will run off the bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the gel is stained in order to be able to see the individual bands of the different proteins. When the gel is stained, the protein distances will be able to be measured and compared. For a detailed procedure, refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.

Nearly 50 years after the first lysozyme crystal structure had been published, the missing piece, the SN2 reaction of the glycosyl-enzyme intermediate, was found. The first lysozyme structure was that of a hen egg white, and it provided a deeper understanding of the mechanism of enzyme reactions. Lysozyme enables the transfer of a glycosyl group to water to occur more quickly. The cleavage of the C-O bond in glycosides without the lysozyme occurs rather slowly.

The shape and magnitude of the UV spectra depends on the composition of amino acid in each protein. Due to the aromatic amino acid residues in the protein, the observed UV absorbance was mainly in the 240 nm to 340 nm region. In Figure 1 to 3, the maximal absorbance of each protein was approximately at 280 nm. The difference in magnitudes of the peak observed was linked to the differences in the amino acid contents in each of the proteins. The peak of lysozyme was greater than those of BSA and gelatin, because lysozyme has a greater number of tryptophan residues. Lysozyme has six tryptophan residues, whereas BSA and gelatin has two and zero, respectively (Department of Chemistry, 2014). Lysozyme has three times more tryptophan residues

The way that the amino acids are arranged makes it specific for only one type of substrate. Once the correct substrate binds to the enzyme there are subtle changes made to the active site. This change is called an induced fit. Then the enzyme converts the substrate into products. Once the products are released, the enzyme returns to its original form.

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

This experiment was conducted to test the properties of enzymes. Enzymes significantly reduce the activation energy reactions by binding to substrate molecules. Enzymes function optimally due to the temperature and pH. At certain temperatures and pH, bonds such as hydrogen and ionic are broken and this allows an enzyme to bind to its substrate. The proteolytic enzyme trypsin was used for this experiment. More about function of this enzyme is included in the lab manual.

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

Student’s attempt to make lysozyme bind tighter to CM Sephadex by starting pH lower than 7 is unsuccessful because the charged groups on the column itself are also titratable groups with specific pKa values. At pH 3 the COO – group of CM column will become protonated and will no longer are able to bind proteins.

Further, since the conformational state of myoglobin can be describe in a two-state thermodynamic model that leads to an equilibrium, it is possible to describe the transition in terms of G as a function of the denaturing agent concentration, as shown in the second plot of Figure 2. In addition, the formulation of a linear perturbation plot permits us to calculate for the Gounfolding of myoglobin in the absence of GuHCl, which was found to be +33.6 kJ/mol as seen in Table 1. On the other hand, the second section of the experiment involves the usage of different concentration of the magic buffer solution, which acts as the denaturing agent for the protein. As illustrated in Figure 3, the absorbance intensity at 409nm was plotted as a function of pH of the protein solution.

Campbell and Farrell define proteins as polymers of amino acids that have been covalently joined through peptide bonds to form amino acid chains (61). A short amino acid chain comprising of thirty amino acids forms a peptide, and a longer chain of amino acids forms a polypeptide or a protein. Each of the amino acids making up a protein, has a fundamental design that comprises of a central carbon or alpha carbon that is bonded to a hydrogen element, an amino grouping, a carboxyl grouping, and a unique side chain or the R-group (Campbell and Farrell 61).

The good results obtained with the epoxy crosslinked enzymes encourages us to make a step forward and implemented the method for magnetic CLEAs. Magnetic CLEAs have the advantage over the CLEAs of being directly removed from the reaction medium with a magnet avoiding centrifugation steps. The procedure was the same as the CLEAs but including amino derivatized magnetic particles to crosslink with the protein, resulting in protein and magnetic particles aggregates. The precipitants selected for this assays were ammonium sulfate as it works for the normal CLEAs and a 50 % PEG 6000 (polyethylene glycol with an average molecular weight of 6000) solution. PEGs are well known protein precipitants, they adsorb water dehydrating the proteins and precipitating

It was first introduced as a concept of “molecular stapler”, where Sortase A was used to covalently bind a tagged green-fluorescent protein (GFP) under native conditions to modified polystyrene beads by incorporation of ligation tags. (Parthasarathy et al., 2007) Sortase A able to bind proteins through covalent bonds by cleaving between theorinine (T) and glycine (G) at ligation sorting motif of LPXTG, where X can be any amino acid residues to generate an intermediate complex of acyl-SrtA. which then reacts with the tri-glycine sequence at N-terminal.