Concept of Biopharming• 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 biopharming’s 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 Creutzfeld–Jakob 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.

Transgenic animals and plants as Bioreactors

• Recombinant-protein synthesis in animal cells has a number of advantages over microbial expression systems, the most important of which is the authentic post-translational modifications that are performed in animal cells.

• However, large scale culture of animal cells is expensive.

• The production of growth hormone in the serum of transgenic mice provided the first evidence that recombinant proteins could be produced, continuously, in the body fluids of animals.

• Secretion of recombinant proteins in mouse milk was reported. This was achieved by joining the transgene to a mammary-specific promoter, such as that from the casein gene.

• The first proteins produced in this way were sheep β-lactoglobulin and human tissue-plasminogen activator (tPA).

Plants as bioreactors

• Plants are a useful alternative to animals for recombinant-protein 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 recombinant-protein 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.

Microbial production of Pharmaceutical compounds

• Human disorders – due to the absence or malfunction of a protein normally synthesized in the body.

• Treatment by supplying the patient with the correct version of the protein, but for this to be possible the relevant protein must be available in relatively large amounts.

• Obtaining sufficient quantities will be a major problem unless donated blood can be used as the source in some cases.

• Animal proteins are used whenever these are effective, but there are not many disorders that can be treated with animal proteins and there is always the possibility of side effects such as an allergenic response.

Microbial production of Insulin

• Insulin, synthesized by the β-cells of the islets of Langerhans in the pancreas, controls the level of glucose in the blood.

• Deficiency leads to Diabetes mellitus.

• Treatment with insulin injections and supplementing the limited amount of hormone synthesized by the patient’s pancreas.

• Traditionally obtained from the pancreas of pigs and cows slaughtered for meat production.

• Although animal insulin is generally satisfactory, problems may arise in its use to treat human diabetes.

• slight differences between the animal and human proteins may lead to side effects in some patients.

• the purification procedures are difficult and potentially dangerous contaminants can not always be completely removed

Lac promoterlacZ’ A gene

Lac promoterlacZ’ B gene

Vector carrying the Artificial A and B genes

met

β –galactosidasesegment

A chain

β –galactosidasesegment

met

B chain

A B

Transformed E. coli synthesizeA and B fusion proteins

Purification of A and B chainsAttach by disulphide bridges

Synthesis of insulin protein

Cyanogen bromide

Cleaved fusion proteins Cleaved fusion proteins

BA

Synthesis of human growth hormone in E. coli

• Somatostatin and somatotropin

• Somatostatin - somatotropin release-inhibiting factor (SRIF)• expressed in the central and peripheral nervous systems, the gut, and other

organs• also inhibit the release of thyroid-stimulating hormone; prolactin; insulin; and

glucagon besides acting as a neurotransmitter and neuromodulator.

• 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.

Lac promoterlacZ’ Artificial somatostatin gene

β –galactosidasesegment

met

Somatostatin fusion protein

Transformation into E. coli

Cyanogen bromide

Cleaved somatostatin

Production of recombinant 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.

Production of recombinant somatotropin cDNA fragmentCodons 0 24 191

HaeIII

0 24 24 191

Discard Retain

Synthetic leader sequence

Expression of somatotropin

Lac promoterlacZ’ somatotropin gene

E. coli transformationSomatotropin issynthesized

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 β-actin and rabbit β-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

Tissue Plasminogen Activator (tPA)

• naturally occurring protease enzyme, helps to dissolve blood clots inside a blood vessel

• Boon for patients suffer from thrombosis

• 1st pharmaceutical product to be produced by mammalian cell culture

• Majority of the natural deaths – due to blockade of cerebral/coronary artery (thrombus)

Production of recombinant tPASynthesize cDNA molecule for tPA

Attached to synthetic plasmid

Introduced into mammalian cells

Cultured and tPA producing cells were selected by using methotrexate to the medium

tPA producing cells were transferred to fermenter

tPA secreted into culture medium is isolated for therapeutic purpose

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 α

Interferon β

Interferon γ

rDNA derived therapeutic agents (approved by FDA) with trade names and their applications in humans

rDNA product Trade name Application/uses

Insulin Humulin Diabetes

Growth hormone Protropin/Humatrope Pituitary dwarfism

Hepatitis B vaccine

Recombinax HB/Engerix B

Hepatitis B

Tissue Plasminogen Activator (tPA)

Activase Myocardial infarction

Factor VIII Kogenate/Recombinate Hemophilia

DNase Pulmozyme Cystic fibrosis

Erythropoietin Epogen Severe anaemia with kidney damage

Some other rDNA derived therapeutic agents approved by USFDA

rDNA product Application/uses

Coagulation factor VIII Hemophilia A

Coagulation factor IX Christmas disease (Hemophilia B)

Interferon α Leukemia

Interferon β Multiple sclerosis

Interferon γ Chronic granulomatous disease

Interleukin 2 Renal cell carcinoma (Kidney cancer)

Interleukin 10 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

high molecular weight molecules – biopolymers (Xanthan gum)– melanin – rubber– polyhydroxyalkanoates etc.