Recombinant DNA Technology: Insulin, Somatropin, and Protein Engineering
Insulin
Insulin is a classic example of a naturally occurring protein produced by recombinant technology. Human insulin was the first recombinant protein commercially produced for therapeutic use. Before the availability of recombinant insulin, diabetic patients relied on insulin purified from the pancreases of pigs and cows, which occasionally resulted in serious immune reactions. Recombinant human insulin has dramatically improved the therapy for diabetes. Human insulin is expressed as a precursor form, single-chain prepro-insulin, which contains extra amino acids that are subsequently cleaved by proteases during the normal processing of this protein hormone. The mature insulin molecule consists of two short polypeptide chains A and B that are linked by two disulfide bonds. Recombinant human insulin was originally produced as the form of proinsulin that subsequently underwent enzymatic cleavage to form the active insulin molecule, but currently, it is produced by expressing A and B chains separately, then refolding them into a mature insulin molecule (Figure 1-1).
Somatropin
Somatropin is the recombinant human growth hormone (rhGH), identical to the pituitary-derived human growth hormone with respect to amino acid sequence (191 amino acids, 22 kDa). Somatropin is synthesized in E. coli as a precursor consisting of the GH molecule conjugated with a secretion signal from an E. coli protein, which directs the precursor to the plasma membrane of E. coli. The signal sequence is then removed, and the GH protein is secreted into the periplasm so that the protein is folded appropriately as it is expressed. Somatropin is indicated for the treatment of growth failure resulting from chronic renal insufficiency or endogenous growth hormone deficiency. Somatropin is also indicated for treatment of short stature associated with Turner’s syndrome.
Recombinant human growth hormone has been investigated in clinical studies for patients with chronic heart failure. Serum levels of insulin-like growth factor (IGF)-I, reflecting endogenous GH secretion, are diminished in relation to the severity of heart failure in patients with dilated cardiomyopathy, and administration of rhGH increases the IGF-I levels, resulting in significant improvement of ejection fraction. In patients with idiopathic dilated cardiomyopathy, rhGH increases myocardial mass and VO2 max, while it reduces the left ventricular chamber size; myocardial sympathetic drive; serum levels of aldosterone; and proinflammatory cytokines including tumor necrosis factor (TNF)-α, its soluble receptors (sTNF-RI, sTNF-RII), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), and soluble Fas/FasL system. However, the results from randomized studies have been conflicting. In some studies, rhGH improved hemodynamics, myocardial energy metabolism, and clinical performance,8–11 whereas other studies showed no benefits in improving clinical status, cardiac function, or neuroendocrine activation in patients with dilated cardiomyopathy, despite a significant increase in left ventricular mass.12,13 These contradictory results may be the result of variable levels of IGF-I response to rhGH administration.14
Recombinant Human Insulin-like Growth Factor-I (rhIGF-I)
Recombinant human insulin-like growth factor (rhIGF)-I, which contains 70 amino acid residues (7.5 kDa), is produced in a variety of expression systems. Endogenous IGF-I may play a pivotal role in compensated heart failure, because the serum levels of IGF-I are elevated in mild to moderate heart failure (NYHA class I and II) but not in severe heart failure (NYHA class III and IV).15 Although it has not been approved for clinical use, rhIGF-I has vasodilatory and positive inotropic effects and has been tested in human patients with heart failure. In healthy individuals, rhIGF-I significantly increases stroke volume, cardiac output, and ejection fraction without increasing heart rate at rest or during exercise.16 In patients with chronic heart failure, rhIGF-I acutely increases stroke volume and cardiac index and decreases pulmonary artery wedge pressure and systemic vascular resistance.17 A number of clinical studies of rhIGF-I and rhGH indicate significant potential of rhIGF-I as a drug to treat chronic heart failure. Further studies to establish long-term efficacy and safety of rhIGF-I are warranted.
Protein Engineering
To modify a protein sequence, researchers substitute, insert, or delete nucleotides in the encoding gene, with the goal of producing a modified protein that is more suitable for an application or purpose than the unmodified protein. Protein engineering is a branch of biotechnology that includes genetic engineering, biotechnology, and biotechnology-related fields. Protein engineering differs from the broader term “targeted mutagenesis” in that it focuses on specific applications. In targeted mutagenesis, also known as site-directed mutagenesis, an alteration is made to a specific site within a gene sequence. It is used in genetic research (Hutchison et al., 1978). Alterations of this nature can be carried out for engineering purposes, such as in protein engineering, or for the purpose of examining the effect of specific mutations in a gene.
Conclusion
Protein engineering is an area of recombinant DNA technology that has experienced rapid growth in recent years. Modifications in genes are expressed as changes in protein conformation that are responsible for the desired properties of the protein. In general, a variety of techniques for the specific engineering of proteins can be divided into two categories: techniques that necessitate extensive prior knowledge of the protein and techniques that help to establish the concept of rational technique of directed evolution that aids in the expression of the progression of natural evolution. Since its inception, protein engineering has thrived in order to produce proteins that have a variety of rewarding applications in industries such as industry, health and medicinal sciences, and ultimately in nanobiotechnology, which is currently the
Chemical Synthesis of DNA
Making DNA chemically rather than biologically was one of the first new technologies to be applied by the biotechnology industry. The ability to make short synthetic stretches of DNA is crucial to using DNA replication in laboratory techniques. DNA polymerase cannot synthesize DNA without a free 3′-OH end to elongate. Therefore, to use DNA polymerase in vitro, the researcher must supply a short primer. Such primers are used to sequence DNA (see later discussion), to amplify DNA with PCR (see later discussion), and even to find genes in library screening. So a short review of how primers are synthesized is included here.
DNA Chemical Synthesis
The synthesis of DNA involves five steps:
Step 1
Nucleosides used for this synthesis are modified with a linking agent at the 3′ hydroxyl group of deoxyribose. The 5′ hydroxyl group of the nucleosides is protected with p-dimethoxytrityl (DMT) ether. The amine groups on the nucleoside’s heterocyclic bases are also protected. The amines of adenine and cytosine bases are protected with benzoyl groups. Guanine’s amine are protected by an isobutyryl group and thymine has no amine groups so protection is not required.
Step 2
Reaction with dichloroacetic acid removes the DMT protecting group from the 5′ hydroxyl of the nucleoside attached to the silica surface. The p-dimethoxytrityl leaving group forms a relatively stable dimethoxytrityl cation which is both tertiary and benzylic. The reaction proceeds rapidly through an SN1 mechanism.
Step 3
The nucleoside attached to the silica surface is then reacted with a protected nucleoside which has a phosphoramidite functional group [R2NP(OR)2] attached to the 3′ hydroxyl group of its deoxyribose moiety. In addition, one of the phosphorus oxygen atoms of the phosphoramidite group is protected with a beta-cyanoethyl group (-OCH2CH2CN). The two nucleosides are coupled in a reaction which uses acetonitrile as a polar aprotic solvent, tetrazole as a heterocyclic amine catalyst, and produces a product with a phosphite functional group [P(OR)3].
Step 4
Next the phosphite product of the previous step is oxidized to a phosphate by reaction with iodine (I2) along with 2,6-dimethylpyridine in aqueous tetrahydrofuran (THF). Additional nucleosides can now be added by repeating the phosphoramidite oligodeoxynucleotide synthesis cycle of (1) DMT deprotection, (2) phosphoramidite coupling, and (3) oxidation to a phosphite.
Step 5
After the oligonucleotide chain of the desired sequence has been made, the final step is the removal of all the protecting groups and the linkage to silica by reaction with aqueous ammonia (NH3).