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It is possible to induce bacterial cells to produce human proteins because the techniques in molecular biology allow the manipulation of DNA wherein one can insert specific DNA segments into the bacterial genome. The bacterial genome is known to be much smaller than the typical eukaryotic genome but it has the ability to perform transcription several times in one run. This process is known as polycistroning transcription. In addition, a bacterial cell carries two DNA strands, the nuclear genome and the plasmid DNA. Plasmid DNAs are closed circular double stranded polynucleotides that carry genes that are also essential in the survival of the bacterial cell in its host (Duarte et al., 2007). An example of a gene that is present in the plasmid DNA is one that codes for antibiotic resistance. The plasmid DNA also has its own origin of replication hence it is possible to make multiple copies of the plasmid DNA depending on the presence of optimal conditions for growth and reproduction.
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In order to make bacterial cells produce human proteins, one should start with a human cell culture that is proliferating at a normal rate. The genome of a human cell contains a gene known as human cytokine (HcytkX) which codes for the human cytokine protein. The initial procedure involves the isolation of the total mRNA content of the cell and this is done using a TRizol reagent which lyses the cell and the reagent differentiates DNA and RNA strands in the solution based on the sedimentation coefficient or mass density of the strands. The differentiation according to mass is done using density gradient centrifugation. Once all the mRNA of the cell is extracted, the specific mRNA that codes for the human cytokine protein can be isolated using a complementary DNA (cDNA) strand which contains the sequence of the human cytokine gene. The cDNA strand will serve as a primer that will identify the actual mRNA strand that is specific for the human cytokine mRNA.
Once the cDNA hybridizes to the human cytokine mRNA strand, it is now possible to generate a DNA copy of the human cytokine mRNA through the process of reverse transcription (Grunstein and Hogness, 1975). An enzyme known as reverse transcription is needed in order to perform this reaction. Once a DNA-RNA hybrid is present, an enzyme known as RNase H is used to clip off or degrade the cDNA probe that was earlier employed. Another enzyme, DNA polymerase is then introduced in order to generate the second copy of the DNA molecule that is complementary to the single strand of DNA.
Once the double-stranded DNA segment that contains the human cytokine gene has been isolated, it is further manipulated using restriction enzymes which are bacterial enzymes that recognize specific DNA sequence motifs. The usual restriction enzymes that are employed in molecular biology experiments are the frequent-cutting restriction enzymes, which cleave the DNA strand at specific 4 to 6 base restriction sites. A specific restriction enzyme will then be introduced to the human cytokine DNA segment is order to generate smaller fragments that are sticky or reactive for reassociation with other DNA segments.
At the same time, bacterial cells such as Escherichia coli (E. coli) should be grown to its exponential growth stage in order to have a sufficient amount of DNA. Once the appropriate amount of bacterial cells is present, the bacterial cells can now be called competent cells because these are ready for DNA manipulation. Plasmid DNA from bacterial cells will be extracted with minipreparation techniques that involve lysing the bacterial cells and centrifuging the cellular solution in order to remove other organelles of the bacterial cell (Birnboim and Doly, 1979). The bacterial plasmid will then be exposed to the same restriction enzyme that was used in the human cytokine DNA segment, also generating sticky ends that are ready to reassociate with other sticky DNA ends (Zhu et al., 2006). The cleaved human cytokine DNA fragments can then be introduced into the bacterial plasmid because both DNA molecules are sticky. The principle of reassociating foreign and host DNA molecules is to employ the same restriction enzyme so that the sticky ends have the same recognition sequences that are complementary to each other. Once the human cytokine DNA fragment is inserted into the plasmid, it is now possible to let the plasmid make more copies of itself inside the bacterial cell.
Bacterial cells multiply very fast and also, the transcription and translation rates of these cells are very short as compared to human cells. Molecular biology techniques allow the manipulation of DNA segments of interest. After incubation of the bacterial cultures that contain plasmids that carry the human cytokine genes, it is then possible to allow the bacterial cells to perform the process of translation, which is the production of protein products based on the transcription results. Translation of the specific human cytokine genes in the plasmid allows that production of human cytokine which can then be collected using isolation techniques. The human cytokine is then further purified using mass chromatographic techniques in order to remove any other unnecessary proteins and other smaller cellular material. The human cytokine protein is then resuspended in a stable buffer such as sterile double distilled water or a buffer such as phosphate buffer in saline solution so that the human cytokine protein remains in its native state. The bottled human cytokine products that are now sold in pharmacies are thus produced through the abovementioned techniques.
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