Essay Sample on Synthesis of Proteins: A Stepwise Process Controlled By Protein Networks

Following the laboratory synthesis of protein, all the stages followed unto the final kind of protein synthesized are to be explained in the paper using the report format. The laboratory synthesis of proteins was, thus, a stepwise process which was highly regulated. In this case, the process was controlled by a complex network of proteins. The proteins synthesized are essential for viability hence the instances if mutation in the situations they are applicable in the real life situations as to be existing in the bodies of living organisms could not be tolerated. It is also because they could adversely affect the fidelity and growth induced by the synthesized proteins. In the lab, there was also the application of the mechanisms to evade the cellular stresses since they could trigger the unwanted repression of the synthesis of proteins. There was also the treatment of the materials to ensure that any viral and bacterial pathogens could be offset as they target the host translation machinery during the protein synthesis. Therefore, the following project design and processes were dully followed in the lab protein synthesis.

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For the project design, there was focus to create NAGZ mutants. There was thus the primer design, as specified above, the PCR, the restriction digestion and litigation, as well as the transformation. The protein purification was also included, to separate it from other proteins, using 6His chromatography. Matrix assisted laser desorption was also employed. The enzymatic characterization involved structural integrity, enzyme kinetics, and temperature denaturation. It was a procedure for the production of a stable functional protein, followed by testing to confirm its functionality.

First, there was the site directed mutagenesis Nagz. The site directed mutagens Nagz was meant to probe the function structure protein relationship. In this case, there was the use of the primer which had a designed restriction site which could in the end ensure a simple and an efficient screening of the proteins being synthesized. There was also the use of a primer wit specific design, in which there was also the undertaking of plating process before the transformation. The prier had 176 Histidine t a Phenylalanine. The name of the primer was c526t_a527_. There was also a specific sequence of primer employed during the synthesis of the protein. The specifications of the primer design in the synthesis of the protein could ensure that there was the availability of the optimal environment for proper output to be realized in the end. The design of the primer also included the use of PCR, which is the Polymerase Chain Reaction. It is a method which is vastly employed in the molecular biology during the making diverse copies of particular segments of proteins, including DNA. The copies of the proteins are amplified in an exponential manner, which in then end produces thousands of the specific protein being synthesized. Duringv5 the process, there was the use of the restriction enzyme DPN1 for the purposes of restriction digestion and ligation. For the case of transformation, there was the use of NEB5a strain.

However, during the process of protein synthesis, there were some potential problems which could hinder the optimal performance in so far as the synthesis of proteins is concerned. In this regard, one of such problems is the improper lab techniques. The laboratory in which there was the synthesis of proteins could not provide an environment that could ensure that the proteins being manufactured are well catered for. The procedures could have been flawed to some extent, which could then lead to the skipping of some processes that were vital in ensuring that the resultant proteins synthesized are well concocted. Additionally, there was an aspect of the low yield of the mutant protein, which could result due to the absence of the enabling environment as is required and provided in a typical lab. Such could then affect the productions in relation to single range or wide range amino acids changes being experienced.

Additionally, there as the potential problem associated with the faulty lab equipment. In this case, the lab synthesis of proteins involved the use of some equipment. In the event that some equipment was faulty, it could adversely affect the provision of an enabling environment for optimal synthesis of the protein. It is because the process in which the protein is being synthesized is enzyme catalyzed, which requires some conditions that make enables the effective synthesis of proteins in the presence of all the provided materials. In this regard, in the event some equipment was faulty, it could adversely affect the protein synthesis process, thus leading to the production of proteins that are not up to the required standard. The equipment could be faulty in the forms of having some cracks which lets air to flow in and out more freely, hence affecting the necessary provided temperature conditions of the reactions taking place in them. Also, the equipment could be faulty in the form the presence of some other chemicals in the equipment, which contaminates them in the process. In this case, the chemicals in them could result from proper washing. They could then react with the materials which have been used in the synthesis of proteins, producing other products which can potentially affect the protein synthesis process.

Also, there was the insufficiency of the resources required in the optimal synthesis of proteins. In this regard, the lab in which the synthesis of the proteins took place had insufficient equipment to be used in the provision of all the conditions necessary for the proper protein synthesis. They include the materials mixed long the reaction process. Also, the resources in the form of temperature regulators, all the required enzymes, alongside the required expertise for proper guidance during the process were also insufficient. Such could then result in the lack proper synthesis of proteins.

For the case of the results obtained from the protein synthesis. There were some graphs constructed to display the result graphically. One if them is the graph of absorbance versus the concentration of nitrophenol. The curve ascended from left to right, which shows a direct proportionality. In this regard, during the protein synthesis process, an increase of nitrophenol concentration leads to a corresponding increase in their absorbance. Since nitrophenol are specific in nature, it shows that a more acidic environment resulting from the increase in nitrophenol concentration favored the enzymatic reactions where the proteins are being synthesized.

In the lab synthesis of proteins, there was the mechanism employed, that could then define the procedure in which the protein was being manufactured. It involved the use of a two - step, double displacement mechanism which involved the formation and breakdown of a glycosyl enzyme intermediate via transition states. During the first step, an enzymic nucleophile attacks the carbon of the sugar while the leaving group departs. The result is the formation of a glycosyl - enzyme intermediate. The second residue assists with the cleavage by acting as a general acid to deliver a proton to the leaving group. In the second step, the residue produced in the first step acts as a base to the incoming molecules of water used in the process. It assists its attacks on the intermediate. The resultant attack by eater aids into the displacement of the enzymic nucleophile, thus releasing the substrate.

There was also the performance of thermal denaturation of mutant NAGZ. In this regard, the results obtained lead to their representation graphically. From the drawn graph, the y axis had the product concentration while the x axis had the temperature readings. The process was pH dependent. The increase in temperature favored the activities of enzymes to some level as shown by the rising curve from left to right, after which denaturation occurred, hence hindering proficient functioning of enzymes, hence making the graph to start dropping from right to left. A high temperature more than 42 denatured the enzymes hence adversely affecting their reactions in the synthesis of proteins.

A graph of absorbance versus concentration of nitrophenol was also constructed. In this case, the rate of absorbance of nitrophenol rises with the increase in the concentration of nitrophenol. The rate of absorbance of nitrophenol also increases with the increase in its concentration. It could thus be concluded that nitrophenol of a higher concentration was necessary in ensuring that the reactions involved in the protein synthesis were at their maximum.

In the lab, there was also the performance of the MULTI - TOF MS analysis. It was performed on the control experiment. In this case, it was found that the plus 1 peak was at around 38,000 ranges. It could thus be seen to verify further the NAGZ protein purification since he mass of the protein NAGZ is at a region of 38kDa. For the case of concentration, there was the construction of a graph which presented the spectrophotometer results. There was the aim to find the concentration and the absorbance whereby the wavelength of 280 nm was recorded, in accordance with Boor's law. The results were represented graphically; the concentration was found to be 0.049148 mmol L, 1.6cmM absorbance, molar absorptivity of 32,555 and path length of 1 cm.

There is also the catalytic mechanism of NAGZ enzyme in operation, which uses a two-step, double displacement mechanism which involves the formation and breakdown of a covalent glycosyl - enzyme intermediate via transition states. The unweaver - Burk plot of the mutated enzyme kinematics shows a direct proportionality, as indicated by a rising curve from left to right. The enzyme kinetics depends on the concentration of nitrophenol which affects absorbance. During the synthesis of proteins, there were the chosen mutation expectations, which include the hypothesis that 176 Histidine is an important part of the active sire as a proton donor/acceptor. If the Histidine at the site 176 is changed to phenylalanine the protein will not function efficiently. Protein purification was also undertaken for the separation of specific proteins from a complex mixture. There was also the hypothesis visualization, using the software for effective exploration of the appearance of the protein. The was highlighting of the active sites of NAGZ H176.

It can be understood that the protein of focus was NAGZ protein, which is the Glycoside Hydrase. It is fund in cytoplasm, with 36 kDA. One of its products, known as the 1,6 - anhydro - N - acetylmuramic, is known to activate beta - lactam antibiotic resistance in gram negative bacteria. It can be observed that new hypothesis got established, that the Arginine at the site 133 is an important binding site. Also, if the Arginine at the site 133 is changed to Alanine then the protein will not function efficiently. The hypotheses confirmed are that if the Arginine at the site 133 is changed to Alanine then catalytic activity is affected negatively. Also, there was the creation of a stable structural purified mutant protein. Finally, the proteins do not react with any substrate. During the experiment, there were the featured sites, including the Aspartic acid (D) 62: Binding site, Arginine (R) 70: Binding site, Arginine (R) 133: Binding site, Histidine (H) 176: Active Site (proton donor/acceptor), and Aspartic Acid (D) 248: Active site (Nucleophile). Finally, the questions which arose, concerning therapeutic uses. They touched on the inhibition of NAGZ and the question: which featured sites have the biggest effect on inhibition.