Proteins are one of the important chemicals in our daily lives which play an important role in our bodies and in the science of chemistry as well. It was first discovered by the Dutch chemist Gerhardus Johannes Mulder in 1838. The proteins basically are the amino acids, and they are linked with the bonding which is called the polypeptide bond. In the bonding of the elements involved are the nitrogen, carbon and oxygen atoms. This paper will discuss about the molecular cloning of protein, its manipulation with respect to DNA, expression in E.coli and its purification.
The molecular cloning of protein refers to the assembling of the DNA molecules with the replication with other organisms. There are seven steps which are involved in the molecular cloning and that is the selection of organism, DNA preparation, preparing of DNA libraries genome sequencing, peptide sequencing and chromosome walking. A detailed planning is done (Doorbar, 2015). It is an extensive process and it involves the study of unlimited fragments of DNA that is derived from any kind of genome. This method has various applications and is applied in biological science. The major application in molecular cloning is the cDNA libraries where the cloning process takes place. The process is such that the DNA to be examined is taken from the organism of interest. Here the DNA is purified in which the proteins are removed and in this way the process goes on (Li, 2017). It is to be noted that the cDNA cloning refers to the cloning representative of the mRNA population and so the precise sequence is specified by the designer. Another important topic is the genome sequencing which deals with the sequencing process of the genes. The process is such that the DNA is examined for the sequencing procedure and this is done at a single time. The main point in this regard is the provision of genetic material that is required for genome sequencing. There are various techniques that plays a vital role in executing the genome sequencing process. In addition to the techniques, the analysis of the genome sequencing provides raw nucleotide sequence of the person’s DNA (Fu, 2014). The analysis include that the genome contains a lot of information and its outcome can be stored electronically but it requires a considerable amount of space and computing power capacity. Now comes the peptide sequencing which involves the determination of peptide amino acid sequence that is obtained from the protein digest in order to analyse the properties if peptides that resides in the protein. There are two different approaches in executing this process (Kanehisa, 2015). The first is the data base search and the other one is the de novo sequencing. The first approach relates to the mass spectra data that is submitted to find a mass spectra data. In this the peptide sequence with a correct match is obtained. Whereas the second approach is all about the fragmentation of ions from a mass spectrum. Chromosome walking is phenomenon of positional clone in which the identification, isolation and particular allele process are executed in the genetic library. Each and every gene is examined repeatedly and so they are mapped for their precise locations in the frame of sequence. It is a very time consuming process and each clone contains a 50 KB information of cosmic library of a DNA (Knowles, 2015).
Manipulation of recombinant DNA
It relates to the alteration of DNA molecules that is subjected to genetic recombination. It involves the study of restrictions of the enzymes, PCR, plasmid vectors and the viral vectors. The phenomena is quite similar to that molecular cloning of protein. The study gives the results in the form of remixing the contents of the DNA in all the associated organisms. It requires a cloning vector and the molecule replicates itself in the living cell (Kumar, 2016). It is to be believed that vectors originate from the plasmids or viruses. They contain the genetic signal for replication process. The method used in this regard is the polymerase chain reaction or PCR. Under this process, the restriction of enzyme is an important topic that includes various steps. The restriction refers to the choice of host programs followed by the cloning vector. Their restriction also reflects to the production of products in a controlled way (Van Veld, 2018). Here the enzymes are directed towards the controlling of the reactions taking place in a controlled environment. It is to be noted that the enzyme is basically a protein that has the ability to carry out multiple reactions (Herce, 2017). A PCR is referred to as a chain reaction in which its main purpose is to carry out the replication process with reference to the specific DNA which is selected by the personals. There are two different methods in which the first one is the replication process that is implemented in a test tube which is free of living cells. This process is also called molecular cloning and the second one is the amplification of the replicated DNA in a current sequence. Similarly a plasmid vector is derived from the viruses and they represent small fragments of DNA for DNA replication through genetic signals. Whereas the viral vectors also possess the same property of plasmid vectors and the similarity exist in their origination (Messori, 2014).
Protein expression in E. coli
This process refers to the plasmid vectors, fusion proteins along with the use of fluorescent markers, antibiotic resistance and the plasmid purification for cell transfections. It holds in significance in expressing the molecules which is required for replication. This study also emphasize on the expansion of cancer cells as they try to replicate the original cells. Here are two main processes are involved which are the transcription and translation (Saladi, 2014). The make use of DNA and mRNA as they replicate into protein molecule. A protein expression is related to the many different forms. It exist in E.coli, followed by the plasmid vectors, fusion proteins fluorescent markers, antibiotic resistance, and plasmid purification for cell transfections. It is to be noted that E.coli is bacteria which possess numerous contributions in different cells \& hormones. In proteins they have a vital role in performing different functions. The fact of the matter is that the protein acts as a gene in E.coli. On the other hand, plasmid vectors also play vital role in interacting with gene production and so they contain essential elements in them. Similarly the fusion proteins is another expression in which it accounts for different matters (Ramos da Silva, 2016). Protein also exist in the form of antibiotic resistance which is another function and it helps in preventing any kind side reactions that may harm the body. The last comes the plasmid purification process and in which the protein aids in the purification by become separated from the rest of the mixture.
This process involves the separation of protein molecules from the complex mixture which normally includes tissues, cells or even the entire organisms. For purification process, it includes chromatography, column purification techniques, crystallography, NMR and the mass spectrometry (Ramos da Silva, 2016). So these are the main methods which are responsible for protein purification process. In all these methods, a porous gel is used like silica gel etc. Similarly the HPLC make sue of high pressure liquid and the proteins are mixed with the gradient of organics solvent in this process (Chilkoti, 2016). One of the important method is the gel electrophoresis where the proteins are purified on the basis of their size. Here the gel is used in this process. The purpose of protein purification considers two different aspects. The first is the preparative aspect and the second one is the analytical purification. In the first one, a bulk quantity of proteins are produced which are in purified form whereas the second one accounts for the purification of small amount of protein which allows the researchers to carry out the task related to quantification, purification along with post-translational modifications. The steps for purification involves extraction step where the proteins are extracted and then the second step is emerges which caters for precipitation & differential solubilisation. Here the proteins are precipitated with ammonium nitrate. The last is the ultra-centrifugations where the proteins are separated from the densities contained in a liquid (Chilkoti, 2016). There are other methods as well that includes gel electrophoreses, crystallography and the mass spectrometry. These are the main techniques which are responsible for protein purifications. In HPLC, a pressure liquid is used for driving the solutes through the columns. Similarly the gel electrophoreses makes use of gel that contains a band which gets separated in the end. The mass spectrometer make use of electric and magnetic field in separating the protein particles from the rest of the solubles.
The overall summary consists of the main definition of proteins which basically are the amino acids, and they contain the formulation which is considered to be the advanced level of chemistry. Their contribution is huge due to their extensive usage in many applications. Such applications included the hydrolysis reactions which gives, oils, fats and the chemical called glycerol which is derived from the glycerine. There are four main topics which makes the protein so important that life is impossible with it. The topics include the molecular cloning, manipulation of DNA, expression of protein and the last is the purification procedure of protein. They contribute towards the replication of DNA and in that it contains a lot of information which can be stored electronically. Such contributions have led to the curing of different diseases and it has opened a new way of research & development. Now considering the functions and industrial applications of proteins followed by the development of the medicines. The proteins ranges from carrying out different reactions happening in our bodies to the developing of the final product in less time. They have a huge variety of industrial application, and the industries they are employed are the production of dairy items, production of oils, beverages and alcohols etc. They also serve as the basis for the medicine industry. They are the main ingredients in developing different medicines for curing of all kinds of diseases. The structure they contain is the form of substrate they can add, rearrange, linked to one another and even they break down itself (Lefort, 2017).
Another application of proteins are the hormones which are regarded as the long-distance chemicals signals by the cells. Their main control is to regulate the growth, development of other glands in our bodies, metabolism and the reproduction (Rose, 2015). The point to be emphasized is that the proteins based hormones are also called the peptide hormones. An example can be explained like insulin which is a peptide hormone that helps in regulating blood glucose levels. There is another application that is related to the development of protein data bank. It is a new development that has been introduced recently and it considers the data storage analysis in which it makes use of X-ray diffraction techniques. Various research has been implemented in which it considers the 3-D spherical data which will contain the proteins and nucleic acids. Through this, the data storage and its reaction has been made easy in all due respects It is also considered to be a waterproof one, and so it ensures the complete protection of data from any kind of losses. In order to implement this innovative terminology, various researches are being conducted, and they are all on full swing. The protein data bank has been observed by the worldwide protein data bank. In the end, it is concluded that the proteins are one of the impact matter in the entire universe. It is considered to be the most versatile, and it has been employed in various applications. New researchers are being conducted to utilize proteins and its associated compounds. It is clear that bin the near future, various devices will employ the use of protein and its derivative in order to cater for future challenges. Therefore, it is clear that proteins have a huge contribution in define the roles where it is about the human body or other scientific research and the corporate sectors as well. Thus the molecular structure of proteins is considered to be the vital element in the chemical field. It has opened a new way of discoveries which will soon be uncovered in the near future.
Chilkoti, 2016. ELASTIN-LIKE POLYPEPTIDES: BIO-INSPIRED SMART POLYMERS FOR PROTEIN PURIFICATION, DRUG DELIVERY AND TISSUE ENGINEERING. Biomaterials from Nature for Advanced Devices and Therapies.
Doorbar, J. E. N. G. H. K. C. a. M. I., 2015. Human papillomavirus molecular biology and disease association. Reviews in medical virology.
Fu, B. S. A. C. C. T. G. P. E. C. A. V. M. a. C. A., 2014. ALMOST: an all atom molecular simulation toolkit for protein structure determination. Journal of Computational Chemistry.
Herce, H. S. D. S. A. L. A. M. F. F. M. K. M. R. S. K. E. L. H. a. C. M., 2017. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nature chemistry.
Kanehisa, M. S. Y. K. M. F. M. a. T. M., 2015. KEGG as a reference resource for gene and protein annotation. Nucleic acids research.
Knowles, M. a. H. C., 2015. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nature Reviews Cancer.
Kumar, S. S. G. a. T. K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution.
Lefort, V. L. J. a. G. O., 2017. SMS: smart model selection in PhyML. Molecular biology and evolution. Molecular biology and evolution.
Li, S. M. Ó. N. J. Y. Y. X. W. M. K. T. D. a. A. Q., 2017. Molecular cloning and functional characterization of a putative Elovl4 gene and its expression in response to dietary fatty acid profiles in orange‐spotted grouper E pinephelus coioides. Aquaculture Research.
Messori, L. a. M. A., 2014. Cisplatin binding to proteins: molecular structure of the ribonuclease A adduct. Inorganic chemistry.
Ramos da Silva, S. a. G. S., 2016. Zika virus: an update on epidemiology, pathology, molecular biology, and animal model. Journal of medical virology.
Rose, A. a. H. P., 2015. NGL Viewer: a web application for molecular visualization. Nucleic acids research.
Saladi, S. C. A. a. C. W., 2014. Statistical Models Robustly Predict Membrane Protein Expression in E. Coli. Biophysical Journal.
Van Veld, P. S. B. F. B. L. F. D. G. R. M. B. a. S. J., 2018. Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In Biomarkers. CRC Press.