Cloning is a term used to describe different methods of duplicating biological materials. Plasmids can be used to ‘clone in a gene’ and then express a given protein in mammalian cells. The type of protein used here is called AWP1 protein which is associated with PRK1. This type of protein is a universally expressed protein and the AWP1 gene is switched on during the early development of a being. The AWP1 may play a regulatory role in the mammalian signal transduction pathways. The AWP1 posses a conserved zf-A20 zinc finger domain at its C-terminal. When it is expressed in COS – 1 cells, the Myc-tagged AWP1 will have an apparent molecular mass which is higher than that which is deduced from its amino acid sequence. These types of proteins are very easy to clone and their nature is very unique. Their structures display momentous information about their functionalities and their roles in the body. Their functionality is attributed to the fact that many protein functional domains are modular. The proteins produced through this process can be used in therapeutics, diagnostics and in other biotechnology processes. These are the driving factors into choosing this type of protein.
The fusion proteins are created from originally coded separate proteins. The single polypeptide will have functional properties which are similar to those of the parent proteins. The naturally occurring fusion proteins are important in the study of cancer where they may function as oncoproteins. Chimeric proteins can also be used in the study of disease development when they are manufactured with toxins or antibodies attached to them.
Proteins usually contain plasmids which have specific components used differently in the purification and expression of the chimeric fusion proteins. Plasmids usually occur naturally in the body cells. They can be covalent, crossed or spherical. They can also be in form of extra-chromosomal replicates and usually inherited and occur naturally in body cells. Plasmid vectors are used for the coding areas. This is procedure is commonly done for the eukaryotic genes. This involves the fusion proteins where the expression vectors help in the programming of the proteins used to carry eukaryotic properties. This process is used with a combination of a tag protein. This process is called purification. During this time, purification and expression of fusion proteins and some specific mechanism of the plasmid (pBudCE4.1) contained in the vector are used to carry out the whole process. Cytomegalovirus plasmid can serve as an early immediate promoter in this process. Elongation factors can also be necessary. Plasmids confer phenotypic characters to their host and commonly found in cells. They serve as supporters for high-level self-regulating proteins appearance.
Plasmids which are used in the purification of proteins are the pUc-family of plasmids which is used in DNA recombinant biotechnology. The pUc-family, carry AmpR gene for the purpose of selection and as well the portion for Lac-Z gene which contain a multiple cloning site (MCS). The plasmid multiple cloning site is for this purpose is usually chosen for the purpose of maintaining the reading frame of Lac-Z. This gene is used to encode the protein beta-galatocinase so that when a foreign DNA is inserted to the MCS, the reading frame of the gene tends to be disrupted. This means there will be the production of a new protein component which is purified.
Patent Claims affecting Sale of the Protein Produced
Products which are intended to be supplied to the users need to be patented. Patents are usually legally imposed and every individual has to follow it to the later. Patents restricts the entry of any Tom, Dick and Harry into a field. It tries to regulate how people get involved in a given kind of undertaking. They protect customers from exploitation which may have been brought about by monopolization of a given industry. The Council for Responsible Genetics has really opposed this idea thus affecting the sale of locally produced proteins. Other claims are that the means of production should be in accordance with the specific regulations set by the law. Some biotechnologists argue that corporations should not be allowed to take or claim possession over specific genes, species and patents whether they have been genetically distorted or occurring naturally. This has also greatly affected the sale of proteins produced.
Other important claims and reasons that can influence the sale of newly produced proteins are truly argumentative. According to many people, patents governing living products and organisms appear decently offensive and the reason they have to be omitted. Because of this, many people do not understand or perceive the patenting of organisms as something that encourages this kind of idea and also make sure someone’s work has not been exploited (8). This will definitely hinder the sale of such locally produced proteins (16). Therefore, it is necessary to note that the above discussed patent claims will definitely have to affect the sale of the newly produced proteins. Before the produced proteins have been patented, it is necessary to make sure these quality issues have been keenly addressed.
The patents that are relevant to each plasmid (pBudCE4.1) component
The CMV promoter is a limited use label license that is covered under U.S Patent No. 5,168,062 and sold for research use only. It is licensed and owned by the University of Iowa Research Foundation, U.S and was filled on 20th February, 2001.
The EF-1(Human Elongation Factor 1α-subunit) Promoter is another limited use label sold under license. It is provided for research purposes only. It’s covered under Patent No. 5,225,348, obtained in Japan and was filled on 15th February, 2002.
There is also the Vectors and Clones Encoding Histidine Hexamer that is licensed under U.S. Patent No. 5,284,933. It is also provided for the purpose of research only and is obtained in Germany. It was filled on 29th June, 2005.
There is a patent that prevents the modifications of Laemmli-type SDS-PAGE, by ensuring maximum separation for the protein analysis. This is the Norvex NurPAGE Gel System.
Country to Manufacture the Proteins: Licences needed
During the manufacture of proteins, several factors have to be taken into consideration. They include: the availability of relevant equipments, the availability of a trained workforce and the legal framework of the country. Lack of relevant equipments may stagnate the whole process of production where as the legal framework can deter a person from undertaking the process of manufacturing proteins. A most suitable place in my own opinion is the United States of America which meets all the requirements mentioned above and its good reputation in the biotechnology field.
There are a number of licenses which need to be purchased in order to carry out the process of manufacturing proteins. Each process of manufacturing has its own license which need to be purchased. The licenses are compulsory and monitored legally. Some of the licenses required include:
- Limited Use Label License which states that all business-related users acquire authority of the relevant patents directly from the right authority,
- Limited Use Label License Bac-to-Bac which warrants the merchandise to be sold under the necessary patent authority,
- Limited Use Label License T7 Expression System which defines and monitors the quality issues to do with the produced proteins,
- Limited Use Label License RNA Transfect, requires that exploits of the products in the combination with the methods used for the beginning need to have patent applications,
- Limited Use Label License Vectors and Clones Encoding Histidine Hexamer which addresses issues related to the materials and products provided for research purposes.
Minimizing Licenses Needed before the Plasmid is used Commercially
In as much as commercialization of products need to be licensed, care should be taken so as to minimize the amount of money spent in obtaining the licenses. Various methods can be used to minimize the number of licenses needed before commercially using the plasmid.
This can be achieved by taking a license that can cater for more than two stages of development. If one takes a license for every stage, then you are likely to spend much more than a person who takes license that can be used for than one stage in the development of the product.
Care should also be taken to ensure that a license does not expire. If a license expires, then one is likely to spend more time renewing it after the expiry date as compared to one who renews a license before the expiry date. Renewing old licenses prevents the acquisition of new licenses. Sometimes it is necessary to produce proteins with lesser restrictions of use. This means that lesser licenses of operation will be required for the protein products.
Consultation with relevant authorities or similar players in the field can also help one to spend less on licenses.
Plasmid use in protein production is very important. Proteins produced will have very many uses in the field of medicine and biotechnology. The patents are very important as they will always protect all the players in the field of biotechnology. License issuance is also an important area as it ensure that the produced items are of the required quality.
List of References.
1. Dower, WJ. A General Approach to the Introduction of Cell Macromolecules. BioTechniques 6, 742-751.
2. Erich, HU. The Difference between Doctors and Nurses. Journal of Human Affairs and Professionalism, 12(2), 34-46.
3. Li, HS. Maximizing Plasmid stability and production of released proteins. Journal of Biotechnology. 3(6,), 123-342.
4. Liao, LM. A transient expression vector for recombinant protein. Journal of Advanced Biotechnology. 2(4), 44-63.
5. Morency, CA. A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression. Biotechniques journal. 5(3); 444-447.
6. Nagata, SK. Characterization of the Human Chromosomal Gene for Polypeptide Chain Elongation Factor-1. Journal of Biol. Chem. 6(4): 51-98.
7. Nelson, JA. Positive Regulation: Short Segment in the Human Cytomegalovirus Major Immediate-Early Gene. Molecular Cell Biology. 7(3): 4125-4129.
8. Roge, JK. Microbial Cell Factories. Journal of Immunology. 2(2), 34-78.
9. Sarah, BD. Gastroenterology Nursing’, Journal of Nursing, 12(3), 34-56.
10. Spring Harbor Laboratory Press; 2004.
11. Storms, RM. Plasmid vectors for protein production and gene expression. Journal of Biotechnology. 3(2), 23-45.
12. Wigler, MH. Transfer of Purified Herpes Virus. Journal of Biology. 1(1), 223-232.