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Knowledge on structure and function of cellular macromolecules Isolation and characterization of genes, cloning a gene and studying its structure and expression through recombinant DNA (rDNA) technology rDNA technology in biological research rDNA technology in pharmaceutical industry
The concept of biopharming is not new. Common medicines, such as codeine, morphine, bulk laxatives, and the anticancer drugs such as taxol and vincristine have long been purified from plants. But biopharmings great promise lies in using genetic modification i.e., techniques to make wild (nontransformed) plants to do drastic new things. Biopharming offers tremendous advantages over traditional methods for producing pharmaceuticals. Great potential for reducing the costs of production. The energy for product synthesis comes from the sun, and the primary raw materials are water and carbon dioxide. To expand production, it is much easier to plant a few additional hectares than to build a new bricks-and-mortar manufacturing facility. Vaccines produced in this way will be designed to be heat-stable so that no refrigeration chain from manufacturer to patient will be required. This would have a great application in developing countries, especially in the tropics and throughout Asia and Africa.
Use of Microbes Earlier days microbes were well exploited among which bacteria are highly exploited. Eg. human growth hormone and insulin. Prior to the advent of genetic engineering, human growth hormone was produced from pituitary glands removed from cadavers. It resulted in recipients contracting CreutzfeldJakob syndrome. The recombinant approach resulted in unlimited supplies of safe material. This safety aspect has been extended to various clotting factors that were originally isolated from blood but now carry the risk of HIV infection. As the methods for cloning genes became more and more sophisticated, an increasing number of lymphokines and cytokines were identified and significant amounts of them produced for the first time.
Use of microbes for production of recombinant therapeutic proteins has several problems The foreign gene may contain sequences that act as termination signals. The codon usage of the gene may not be ideal for translation in bacterial system (Codon bias). Lack of post translational modification and correct folding of the human recombinant proteins in microbial system. Degradation of recombinant proteins since it is someway recognized as foreign protein in bacteria.
Plants as bioreactors
Plants are a useful alternative to animals for recombinantprotein production because they are inexpensive Therefore, there is much interest in using plants as production systems for the synthesis of recombinant proteins and other speciality chemicals. There is some concern that therapeutic molecules produced in animal expression systems could be contaminated with small quantities of endogenous viruses or prions, a risk factor that is absent from plants. Furthermore, plants carry out very similar post-translational modification reactions to animal cells, with only minor differences in glycosylation patterns. Thus plants are quite suitable for the production of recombinant human proteins for therapeutic use.
The first such report was the expression of human growth hormone, as a fusion with the Agrobacterium Nopaline synthase enzyme, in transgenic tobacco and sunflower. Tobacco has been the most frequently used host for recombinantprotein expression although edible crops, such as rice, are now becoming popular, since recombinant proteins produced in such crops could in principle be administered orally without purification. The expression of human antibodies in plants has particular relevance in this context, because the consumption of plant material containing recombinant antibodies could provide passive immunity (i.e. immunity brought about without stimulating the host immune system). Antibody production in plants was first demonstrated by Hiatt and During team, who expressed full-size immunoglobulins in tobacco leaves. Since then, many different types of antibody have been expressed in plants, predominantly tobacco, including full-size immunoglobulins, Fab fragments and single-chain Fv fragments (scFvs).
A fully humanized antibody against herpes simplex virus-2 (HSV-2) has been expressed in soybean. Even secretory IgA (sIgA) antibodies, which have four separate polypeptide components, have been successfully expressed in transgenic tobacco plants. Plants producing recombinant sIgA against the oral pathogen Streptococcus mutans have been generated, and these plant-derived antibodies (plantibodies) have recently been commercially produced as the drug CaroRxTM, marketed by Planet Biotechnology Inc.
A gene
B gene
B B chain
Agromegaly (uncontrolled bone growth) and dwarfism. Somatostatin was the first human protein to be synthesized in E. coli. Somatostatin - very short protein, only 14 amino acids in length, it was ideally suited for artificial gene synthesis. Strategy - insertion of the artificial gene into a lacZ reading frame of the pBR 322 vector, synthesis of a fusion protein and cleavage with cyanogens bromide.
Cyanogen bromide
Cleaved somatostatin
Somatotropin Somatotropin -191 amino acids in length, equivalent to almost 600 bp, Combination of artificial gene synthesis and cDNA cloning was used to obtain a Somatotropin-producing E. coli strain. mRNA was obtained from the pituitarygland and a cDNA library was prepared. The Somatotropin cDNA turned out to have a unique site for the restriction endonucleases HaeIII, which cuts the gene into two segments. The longer segment, consisting of codons 24 to 191, was retained for use in construction of the recombinant plasmid. The smaller segment was replaced by an artificial DNA molecule that reproduced the start of the Somatotropin gene and provided the correct signals for translation in E. coli. The modified gene was then ligated into an expression vector carrying the lac promoter.
Retain
pression of somatotropin
Synthetic leader lacZ sequence Lac promoter somatotropin gene
Recombinant Factor VIII Central role in blood clotting. Recombinant pharmaceutical protein produced in eukaryotic cells Haemophilia Treatment - injection of purified Factor VIII protein, obtained from human blood provided by donors. Purification is a complex procedure and the treatment is very expensive Difficult to remove the virus particles that are present in the blood. Hepatitis and AIDS can and have been passed on to haemophiliacs via Factor VIII injections. Recombinant factor VIII, free from contamination problems, would be significant achievement for biotechnology.
The factor VIII gene is very large, Over 186 kb in length, and is split into 26 exons and 25 introns. The mRNA codes for a large polypeptide (2351 amino acids) which undergoes a complex series of post-translational processing events, eventually resulting in a dimeric protein consisting of a large subunit, derived from the upstream region of the initial polypeptide and a small subunit from the downstream segment.
The two subunit contain a total of 17 disulphide bonds and a number of glycosylated sites. It is not possible to synthesize an active version in E. coli. Most attempts made on mammalian cells.
First, entire cDNA was cloned in hamster cells, but yields of protein were disappointingly low.
Because of the failure in post-translational events, (do not convert the entire initial product into an active form limiting the overall yield). As an alternative, two separate fragments from the cDNA were used, one fragment coding for the large subunit polypeptide, the second for the small subunit. Each cDNA fragment was ligated into an expression vector, downstream of the Ag promoter (a hybrid between the chicken -actin and rabbit -globin sequences) and upstream of a polyadenylation signal from SV40 virus. The plasmid was introduced into a hamster cell line and recombinant protein obtained. The yields were over ten times greater than those from cells containing the complete cDNA and the resulting factor VIII protein was indistinguishable in terms of function from the native form
Cultured and tPA producing cells were selected by using methotrexate to the medium
Synthesis of other recombinant human proteins Interferon and interleukins - cancer therapy. Serum albumin, are more easily obtained, but are needed in such large quantities that production in microorganisms is still a more attractive option. Erythropoietin hormone synthesized by the kidney stimulate the stem cells of bone marrow to produce mature erythrocytes
Interferon Antiviral substance First line of defense against viral attacks Glycoprotein in nature Containing a group of > 20 substances with mol. Wt of 20000 - 30000 daltons
Interferon Interferon Interferon
rDNA derived therapeutic agents (approved by FDA) with trade names and their applications in humans rDNA product Insulin Growth hormone Trade name Humulin Protropin/Humatrope Application/uses Diabetes Pituitary dwarfism Hepatitis B Myocardial infarction
Hepatitis B vaccine Recombinax HB/ Engerix B Tissue Plasminogen Activase Activator (tPA)
Kogenate/Recombinate Hemophilia Pulmozyme Epogen Cystic fibrosis Severe anaemia with kidney damage
Some other rDNA derived therapeutic agents approved by USFDA rDNA product Coagulation factor VIII Coagulation factor IX Interferon Interferon Interferon Interleukin 2 Interleukin 10 Application/uses Hemophilia A Christmas disease (Hemophilia B) Leukemia Multiple sclerosis Chronic granulomatous disease Renal cell carcinoma (Kidney cancer) Thrombocytopenia (few platelets in blood)
Restriction endonucleases
> 500 different restriction endonucleases Synthesized by a wide range of microorganisms and for each organism, a detailed fermentation protocol, has to be developed and optimized To avoid having to maintain a large number of different microorganisms, stock a very wide range of medium components, design several different fermenters and spend an inordinate amount of time developing optimal growth conditions for a large number of different organisms, researchers often clone restriction enzyme gene into E. coli. Because it is easy to standardize the conditions and E. coli cells grow rapidly to high cell densities and can be engineered to significantly over express the target restriction enzymes.
However, host organism is dramatically affected by the production or presence of a heterologous protein. Over expression of heterologous protein may drain the host organism of important metabolic resources and this may affect its growth or sometimes it may be lethal to the host. Eg. There is a possibility to digest the host DNA by the heterologous restriction enzyme unless a protection mechanism is present.
Microbial system has also been used to synthesize several industrially important low molecular weight molecules
L-ascorbic acid (Vitamin C) indigo amino acids antibiotics and biopolymers (Xanthan gum) melanin rubber polyhydroxyalkanoates etc.