Biopharming Training
Biopharming Training
Biopharming Training
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
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 virus2 (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.
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.
Lac promoter
lacZ
A gene
Lac promoter
lacZ
B gene
A A chain
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
Expression of somatotropin
Synthetic leader sequence lacZ somatotropin gene E. coli transformation Somatotropin is synthesized
Lac promoter
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
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 Trade name Humulin Application/uses Diabetes
Growth hormone
Hepatitis B vaccine Tissue Plasminogen Activator (tPA)
Protropin/Humatrope
Recombinax HB/ Engerix B Activase
Pituitary dwarfism
Hepatitis B Myocardial infarction
Factor VIII
DNase Erythropoietin
Kogenate/Recombinate Hemophilia
Pulmozyme Epogen Cystic fibrosis Severe anaemia with kidney damage
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
Transgenic animals
Gene transfer to animal cells has been practiced for the past 40 years. Techniques are available for the introduction of DNA into many different cell types Animal cells - advantageous for the production of recombinant animal proteins (authentic posttranslational modifications) that are not carried out by bacterial cells and fungi. Cell cultures - commercial scale to synthesize products
Intense research - efficient vector systems and transformation methods for animal cells.
Gene-transfer strategies
(1) Direct DNA transfer - the physical introduction of foreign DNA directly into the cell.
microinjection - in cultured cells bombardment with tiny DNA-coated metal particles - for cells in vivo.
(2) Transfection - chemical and physical, which can be used to persuade cells to take up DNA from their surroundings. (3) Transduction: Packaging the DNA inside an animal virus
Transformation can be transient or stable, depending on how long the foreign DNA persists in the cell.
Proteins with therapeutic and industrial value have been produce (not commercialized) in the milk of transgenic animals
Protein Antithrombin III Factor VIII and IX CFTR (Cystic Fibrosis Transmembrane conductance regulator) Lactoferrin Alpha 1 anti trypsin Lysostaphin Spider silk protein Animal Goat Goat, Pig, Sheep Sheep Use Reduce the amount of blood needed in some surgeries Treatment of Hemophila Treatment of Cystic Fibrosis
Natural antibiotic and used in coronary surgery Treatment of Cystic Fibrosis and emphysema Antibacterial compound that prevents mastitis in cows Production of ultra strong and light weight industrial materials
Transgenic animals:
Animals which have been genetically engineered to contain one or more genes from an exogenous source. Transgenes are integrated into the genome. Transgenes can be transmitted through germline to progeny. First transgenic animal produced = Founder Animal
Mouse animal of choice for transgenic expt. Easily handled and researcher friendly Transgenic mice contributed
Understanding of
Mol. Biol Genetics Immunology Cancer studies Animal models for human genetic diseases
Retroviral vector
Transfer small pieces of DNA (8kb) Not suitable for large DNA Risk of losing regulatory sequences Risk of retroviral contamination A commercial product is to be synthesized by the transgenic organism or the transgenic organism to be used as food, it is absolutely necessary that there be no retroviral contamination.
Cleavage stage embryos (eight-cell stage) infected with defective retrovirus carrying a transgene Implanted females (foster mothers)
Transgenic pups
Matings are carried out to determine which pups have the transgene in their germ line cells. Transgenic lines can be established from these founder transgenic animals
Microinjection method
(Super ovulated female) Young virgin female mice (4-5 weeks) FSH (pregnant mares serum) 2 days latter human chorionic gonadotropin Produce 30 35 eggs
Transgene may not be expressed / over expressed / under expressed and affect normal physiology
Time consuming Costly Labour intensive
Cells from the blastocyst stage of a developing mouse embryo can proliferate in cell culture and have the capability of differentiating into all other cell typesincluding germ line cells- after they are reintroduced into another blastocyst embryo (Pluripotency).
Pluripotency in the broad sense refers to "having more than one potential outcome".
Pluripotent stem cells can give rise to any fetal or adult cell type.
Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts.
1. Make your DNA Using recombinant DNA methods, DNA containing the structural gene, vector DNA, Promoter and enhancer sequences to enable the gene to be expressed by host cells
2. Transform ES cells in culture Expose the cultured cells to the DNA so that some will incorporate it. 3. Inject these cells into the inner cell mass (ICM) of mouse blastocysts 4. Embryo transfer(Implantation) Prepare a pseudopregnant mouse (to make uterus receptive). Transfer the embryos into her uterus. develop into healthy pups (no more than one-third will). 5. Test her offspring Remove a small piece of tissue from the tail and examine its DNA for the desired gene. 6. Establish a transgenic strain Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene. Mating these will found the transgenic strain.
Transgenic sheep overproducing growth hormone susceptible to infection, infertile and tend to die at early stage
Due to ineffective control of gene regulation Resistance to diseases, enhancing the milk production, production of commercial and pharmaceutical compounds
Transgenic cattle
Mammary gland of the dairy cattle- ideal bioreactor Transgenic cow over expressed K - casein transgene milk with higher content of casein Lactase transgene lactose free milk lactose intolerant people Resistance to viral, bacterial and parasitic diseases
Mature oozytes are collected from cows and fertilized with semen collected from a bull in vitro.
Fertilized oozytes are centrifuged to settle the yolk at one pole of the oozytes
Foreign gene is microinjected into male pronucleus in the oozytes Oozytes grown in vitro till blastocyst stage Implanted in uterus of foster mother
Transgenic sheep
Mostly involve development of mammary glands as bioreactors for the production of proteins for pharmaceutical use. Eg. 1-antitrypsin, Factor IX 1-antitrypsin is fused with -lactoglobin promoter microinjected into male pronucleus of fertilized egg. 1-35 g of 1-antitrypsin per litre of milk Gene for factor IX Isolated and purified from milk to treat haemophilia
Transgenic sheep
Keratin wool protein, highly cross linked disulfide bridges Cysteine is required in large quantities - quality wool Cysteine supply inadequate Microbes harbouring in the rumen utilize it and release in the form of sulfide Transgenic sheep containing bacterial genes for synthesis of cysteine
Two enzymes , synthesized by the transgenes trap the H2S and liberate in the intestine to produce the cysteine
Good supply of cysteine to the sheep improves the quality and quantity of wool
Transgenic goats
Transgenic goats with tPA gene produces tissue Plasminogen activator in milk
Transgenic pigs
Gene for Factor VIII introduced into zygote of pig by microinjection
Xenotransplantation
Transplantation of animal organs in human system Human organs such as liver, pancreas, kidney and lungs great demand for transplantation surgery Shortage can be overcome by developing them in mammal Pig favourite animal for harvesting human organs But human body produced antibodies against pig organs reject the transplants Hyperacute rejection Imutran Company (USA) produced transgenic pig (Astrid) by microinjecting genes for human immune system into male pronucleus of the zygote
Transgenic rabbits
Interleukin-2 gene along with -casein promoter
Transgenic chickens
Complicated Fertilization several sperms enter the ovum instead of one (contrast to mammals) Identification of male pronuclei that will fuse with female pronuclei is quite difficult ES cells have not been identified in chickens The blastoderm cells can be removed from a donor chicken and are transfected with transgenes Modified blastoderm cells reintroduced into subgerminal space of irradiated blastoderm of freshly laid eggs Transgenic lines are produced Transgenesis for low fat and cholesterol, high protein containing eggs Resistance to viral and bacterial diseases , Production of pharmaceutical proteins Transgenic chickens resistant to Avian Leukosis virus (ALV)
Rabbits
Mouse Mouse
glucosidase
Urokinase Immunoglobulins
Vaccines
Conventional vaccines
Recombinant vaccines
Live vaccines
Inactivated pathogen
DNA Vaccines
Polypeptide
Conventional vaccines
consist of whole pathogenic organisms (bacterial / viral vaccines), which may be either killed or live (attenuated) (live vaccines) (most viral vaccines)
Limitations:
In many cases live vaccine have to be used, killed pathogen vaccines are ineffective Live vaccines are generally based on cultured animal cells and expensive tissue culture set up is essential Live vaccines are heat labile Risk of disease development due to occasional presence of active virus particle or reversion to virulence
Limitations:
Cost is higher due to steps involved in purification and vaccine preparation Many of isolated antigens are poorly immunogenic
Recombinant vaccines
Contains either a protein or a gene encoding a protein of a pathogen origin that is immunogenic and critical to pathogen function; the vaccine is produced using recombinant DNA technology.
Vaccines based on recombinant proteins are called as sub unit vaccines.
Steps involved: Identification of protein that is immunogenic and critical for the pathogen Gene encoding for the protein is identified isolated Gene is inserted into a expression vector and introduced in to suitable host where it produces large quantity Protein isolated and purified from the host cells. It is used for the preparation of vaccine.
Structure and functionality of given protein is determined by its sequence of amino acids, which, in turn, determines its three dimensional confrontation / structure.
Internal bonds (S and H bonds) among the amino acids give the proteins its final shape and form.
Complex proteins undergo further processing such as phosphorylation and glycosolation, which modify proteins functions. Information stored in DNA directs the protein synthesizing machinery of the cell to produce specific protein required for its structure, function and metabolism. Since proteins play critical roles in cell biology, they have many therapeutic uses in preventing and curing diseases and disorders.
Short peptide chains can be synthesized chemically whereas large peptides in living cells.
DNA that encodes the instructions for producing desired proteins is inserted into cells and as the cells grow they synthesize the proteins and subsequently they are harvested and purified.
Therapeutic proteins
Antibodies Passive immunization an immune response that results from injecting another organisms antibodies into organism that is being challenged by the pathogen Passive immunization using MAB are largest category of biotech derived drugs. In passive immunization, rather than injecting an antigen and inducing the body to produce antibodies against it, an antibody targetted towards a specific antigen is administered as a therapeutic. Eg. Multiple doses of Herceptin against breast cancer Antibody therapes are available for lymphoma, rheumatoid arthritis, respiratory synchytial virus Clinical trials are underway.
Antibodies produced from transgenic plants are used for the treatment of
Dental caries Rheumatoid arthritis Cholera E. coli diarrohea Malaria Certain cancers HIV Norwalk virus Rhino virus Influenza Hepatitis B Herpes simplex virus
Vaccines
Protein antigens from various pathogens have been expressed in plants and used to produce immune responses resulting in protection against diseases. Plant derived vaccines against Vibrio cholerae, enterotoxigenic E. coli, Hepatitis B virus, Norwalk virus, rabies virus, human cytomegalo virus, respiratory synchytial virus Insulin expression in plants produceda vaccine useful for protection against insulin dependent auto immune Mellitus diabetes Antigen specific to an individual patient tumor are expressed in tobacco, harvested, purified and administered to the patient.This entire process will take place as little as 4 weeks compared to 9 months for the same process in mammalian cell culture.
Plant derived antigens purified and used as injectable vaccines. Oral delivery of these vaccines with in foods also successful.
Edible vaccines
Low cost delivery mechanism for immunisation No need for injection, sterile needles, and refrigeration Edible vaccines successfully immunized test animals against enterotoxigenic E. coli, V. cholerae, hepattitis B, norwalk virus rabies virus etc. Concentration of vaccine protein in edible vaccine is relatively low. Research is underway to increase them in targeted sites. Eg. Potato, Tomato, banana and carrot Potato cooking inactivate the vaccine. Tomato and banana short storage life Carrot few storage problems, can be eaten raw. (hepatitis B vaccine)
Challenges: Standardization of expression Dosage level and immune responses Distributed through health service channels Decrease the cost of immunisation in developing countries Eg. Golden rice program
2) Derived Products
Bio-plastics - PHAs (polyhydroxyalkanoates, chemically related to polyesters).
Nutraceuticals: Antigens (vaccines) (Active Macro: Carbohydrates, Fats immunity) Micro: Vitamins, co-factors, minerals, Phytochemicals: carotenoids (betaStructural: proteins, peptides, carotene, lycopene, lutein), flavonoids hormones, (interleukins, (quercetin, kaempferol, allicin), interferons and colony stimulating isoflavones (phytoestrogens factors) genistein and daidzein), isothiocyanates (glucosinolates, Enzymes: food, feed, industrial, indoles, and sulforaphane), phenolics therapeutic, diagnostic, cosmetic (reservatrol, catechin), tannins Anti-disease therapeutics: Factor VII, Enzyme inhibitors Non-nutrient phytochemicals: fragrances, flavors Fibres: polymers, lignins
>100 million children suffer from the problem For many countries, the infrastructure doesnt exist to deliver vitamin pills Improved vitamin A content in widely consumed crops an attractive alternative
Phytoene
Phytoene desaturase
-carotene desaturase
Lycopene
Lycopene-beta-cyclase
Normal Vitamin A Deficient Rice
Phytoene
-carotene desaturase
Lycopene
Daffodil gene Golden Rice
Lycopene-beta-cyclase
Transformation Cassettes
Contains 1. Gene of interest The coding region and its controlling elements 2. Selectable marker Distinguishes transformed/untransformed plants 3. Insertion sequences Aids Agrobacterium insertion
Transformation Steps
Prepare tissue for transformation
Tissue must be capable of developing into normal plants Leaf, germinating seed, immature embryos
Introduce DNA
Agrobacterium or gene gun
Non-transgenics
Transgenics