Analysis Of Recombinant Proteins Biology Essay


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Recombinant protein is an artificial protein that is derived from recombinant DNA technique. Recombination is the process of combination of genes to form a new strand of DNA. Recombinant DNA technology was firstly described by several scientists, such as David A. Jackson, Stanley N. Cohen in 1972 to 1974. This technique is done by selecting of a target DNA sequence and then inserting it to a vector that has the ability to self-reproduce. The widely used vector is plasmid of Escherichia coli (Choi, Keum, & Lee, 2006). Plasmids are circular forms of double strand DNA which have advantages of having self-replication independently of the chromosomal DNA, easily manipulate and rapid growth properties. The first step is to select a part of DNA, i.e. the gene of interest. The second step is cut this gene with restriction enzymes and then insert into the plasmid, after that, the bacteria reproduce together with the plasmid containing that gene. The inserted gene from one organism into another organism is so called recombinant DNA (Cohen, Chang, Boyer, & Helling, 1973). The recombinant DNA inside the Escherichia coli undergoes transcription, which produces mRNA template; followed by translation, which in turn produces the recombinant proteins, and finally creating the desired protein. So, recombinant technology is the process involving in the formation of recombinant protein (Demain & Vaishnav, 2009). Recombinant protein plays an important role of creation of therapeutic agents, such as interferons, insulin, erythropoietin, factor VIII, that can modify and repair genetic errors, destroy cancer cell, treat immune system and disorders (Frost, 2005). For instance, insulin, a protein hormone produced by recombinant technology can be utilized in treating patients with diabetes.

Insulin is a hormone produced by the beta cell of islets of Langerhans in the pancreas. The most important function of insulin is control of glucose concentration in the blood when flowing through the pancreas. An increase in blood glucose concentration stimulates insulin secretion, whereas a decrease inhibits secretion. After a meal, rise in plasma glucose concentration stimulates insulin secretion, and the insulin stimulates entry of glucose into muscle and adipose tissue, as well as uptake of glucose by the liver. These effects reduce the blood concentration of glucose, thus removing the stimulus for insulin secretion, which goes back to its previous level (Vander, Sherman, & Luciano, 1994, p.608).

Diabetes can be due to a deficiency of insulin. In insulin-dependent diabetes mellitus (type 1), the hormone may completely absent from the islet of Langerhans and the plasma, so therapy with insulin is essential. In the past, insulin was extracted directly from the pancreas of cattle and pigs, but nowadays human insulin is used. Human insulin has an advantage of not causing immune response by the body, so it will cause less reaction at the injection site and less insulin may be required (Schernthaner G, 1993). But how human insulin can be synthesized in large amount? This can be achieved by using recombinant DNA technology.

Before synthesizing human insulin, firstly we must know the chemical composition of this protein. Insulin is a small and simple protein. It consists of 51 amino acids, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. The two chains are linked by a disulfide bond (Olsen, Ludvigsen, & Kaarsholm, 1998). The genetic code for insulin is found in the DNA at the short arm of the 11th chromosome (Harper, Ullrich, & Saunders, 1981).

Recombinant insulin

There are five steps that must be carried out in order to successfully synthesize human insulin. The first step is to obtain the gene for insulin from human DNA. This is done by isolating mRNA rather than DNA. One reason for this is that mRNA transcribed from a particular gene is more abundant than the gene itself. Then reverse transcriptase uses the mRNA as a template and transcript a complementary strand of DNA, and then converted into double-stranded DNA molecules by DNA polymerase. The second step is to insert the gene into bacterial cells (plasmids). This is done by restriction endonucleases, the enzymes cut double-stranded DNA in a way to produce sticky ends which can be joined by base-pairing of the target DNA. Then, using another enzyme called DNA ligase, which is covalently bond the two DNA fragments, producing a new molecule of DNA called recombinant DNA. The third step is to select bacterial cells that have the human insulin gene. Because of the plasmid vectors we used contain at least one gene for antibiotic resistance. So, we can make use of this property to select bacterial cells containing resistance gene together with human insulin gene which can grow in the presence of the antibiotic medium. Although the bacterial cells can synthesize insulin gene, it is regarded as a foreign gene. The subsequent step is to induce the expression of insulin in bacterial cells. This can be done by using a plasmid expression vector to drive the production of foreign proteins. This plasmid contains control sequences that can initialize transcription and translation no matter what gene is inserted into the plasmid. However, eukaryotic cells often modify proteins after they are synthesized. For insulin, it is firstly synthesized in the beta cells of pancreas in an inactive form, called preproinsulin, which is a single long protein chain with the A and B chains still connected. In order to change to an active form of protein, post-translational modification occurs, this includes leader sequence is cut and folds into a stable conformation with disulfide bonds to form a molecule called proinsulin. After that, the connecting sequence is cut to form the mature insulin molecule, that is, with A and B chain joins together with two disulfide bonds. Thus, if the gene for insulin, which obtained from mRNA, is cloned into bacterial cells and expressed as protein, the bacterial cells will produce the inactive preproinsulin, rather than active insulin. To solve this problem, chemicals synthesize of each chain of insulin have been introduced, provided that the exact DNA sequence of each chain is known, so that each chain can be separately cloned, and the products of each cloning are then mixed and joined by disulfide bonds to give rise to active insulin. Another way to produce human insulin can be started from proinsulin, after cloning, the connecting sequence between the A and B chains is cut away with an enzyme and the active form of insulin is then formed (Bourgaize, Jewell, & Buiser, 2000, p.144). The final step is to purify the product (insulin) away from other contaminant such as the bacterial proteins. Purification can be done through chromatography, or other techniques such as X-ray crystallography), according to the charge, size, and affinity to water of the molecule (Kim et al., 2004).

The use of recombinant insulin

The way of use of recombinant insulin can be improved by changing its amino acid sequence and creating analogues, such as insulin aspart and insulin glargine. Insulin aspart is a fast response analogue, structural different only on the substitution of aspartic acid for proline at position 28 on the B chain (Tamas et al., 2001). Insulin aspart can increase in serum concentration 2.3 times higher and in less than half the time than human insulin (Kaku, Matsuda, Urae, & Irie, 2000). On the other hand, insulin glargine is a long-acting analogue, structural different from human insulin by 3 amino acids, in which asparagines at position 21 of A chain is replaced by glycine and 2 arginines are added to the carboxyl terminal at position 31 and 32 of the B chain. This can shift the isoelectric point from pH5.4 to pH7.0, and making relatively less soluble at the injection site (Wang, Carabino, & Vergara, 2003). Consequently, body absorption of insulin glargine becomes delay and prolongs the duration of effect in order to provide constant supply of insulin over 24 hours after each injection (Campbell, White, Levien, & Baker, 2001).

Abuse of recombinant insulin

Insulin can be abused (doping) by professional athletes because it may improve performance by increasing protein synthesis and glucose uptake and storage (Erotokritou & Holt, 2010). On the other hand, insulin dependent diabetics have an increased incidence of a psychiatric disorder such as depression, anxiety, or euphoria. Even though, some of individuals who are not diabetic may inject insulin to induce euphoria. Nowadays, the majority of insulin dependent patients are treated with recombinant human insulin; however hypoglycaemia may appear in those patients (Graham, Evans, Davies, & Baker, 2008). Although the recombinant human insulin is a successful genetic product for insulin dependent patients, the use of such product should be carefully monitored. Insulin derivatives must continue to be evaluated in order to ensure their proper usage.

Detection method of recombinant insulin

As insulin regulates glucose metabolism in our body, we can measure blood glucose level in order to monitor diabetic patients of proper use of insulin analogues. On the other hand, for insulin misuse in sport, we can measure insulin and C-peptide (connecting peptide, the by-product of the conversion of proinsulin to insulin) in serum as indirect markers by immunoassays such as Enzyme Amplified Sensitivity Immunoassy (EASIA) (Abellan et al., 2009).

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