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From Gene to BioproductsFrom Gene to From Gene to BioproductsBioproducts

Biofactories in the Biotechnology Industry

BiofactoriesBiofactories in the in the Biotechnology IndustryBiotechnology Industry

Bioprocess Engineering WorkshopProf. S. T. Yang

Dept. Chemical & Biomolecular EngThe Ohio State University

OUTLINEOUTLINEOUTLINE• Introduction - Biotechnology• Biomolecules• From Gene to Product (Protein)• Recombinant DNA Technology

Biotechnology Pharmaceuticalsdrugs, healthcare

Human

Diagnostics

Biomedical artificial organs, body parts

Agriculture

Plant tissue cultures,Transgenic plants

Transgenic animals

IndustrialBiochemicals

Environmentpollution control

AAPPPPLLIICCAATTIIOONNSS

Industrial Markets for BiotechnologyIndustrial Markets for Biotechnology

• Pharmaceutical industry - over $400 billion• Agriculture and food industry

– transgenic crops, animals– recombinant bovine somatotropin (BSA)

• Chemical industry – over $2 trillion on sales worldwide– commodity chemicals, specialty chemicals, consumer

care products, and pharmaceuticals; plastics/polymers – uses nearly $24 billion worth of hydrocarbon feedstocks

annually in the US. • Fuel and energy

– 4.5 billion gallons of ethanol from corn in 2006– 75 million gallons of biodiesel from soybean oil in 2005

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BiotechnologyBiotechnology

What is biotechnology?

“Biotechnology, broadly defined, includes any technique that uses living organisms (or parts of organisms) to make or modify products, to improve plants or animals, or to

develop microorganisms for specific uses.”-- Office of Technology Assessment, 1984

Biotechnology is “the integrated use of biochemistry, microbiology, and engineering sciences in order to achieve technological (industrial) applications of the capabilities of microorganisms, cultured tissue cells, and parts thereof”

-- European Federat ion o f Biotechnology, 1985DNA account for about 0.25% cell weight for a typical mammalian cell and 1% for a typical bacterial cell.

CellCell

BiomoleculesBiomolecules

• Carbohydrates

• Proteins

• Lipids

• Nucleic acids

CarbohydratesCarbohydrates

• Contains oxygen, hydrogen and carbon atoms, and no others. Some not

• (CH2O)n or derivations

• Function as storage and transport form of energy

• Classified based on the number or sugar units: Monosaccharide, Disaccharides, Oligosaccharides, and Polysaccharides

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Protein (Polypeptides)Protein (Polypeptides)

• Discovered in 1838 by Jons Berzelius• From Greek, protas: of primary importance• Consists of amino acids arranged in a

linear chain linked by peptide bonds• 20 standard amino acids• Plants and microorganisms can synthesize

all the 20 a.a., animal cannot• Essential amino acids from diet

SizesSizes

• Total molecular mass in daltons or kDa

• Yeast proteins average 466 a.a. and 53 kDa in mass.

• The largest known protein are the titins, a component of muscle sarcomere, MM of 3,000 kDa and 27,000 a.a.

ClassificationClassification

• Globular proteins– Most are soluble – Mostly enzymes, antibodies, hormones

• Fibrous proteins– Structural

• Membrane proteins– Mostly in cell membranes– Receptors – Channels

Functions of ProteinsFunctions of Proteins

• Enzymes: catalyze biochemical reactions, metabolism and biosynthesis

• Cell cytoskeleton, i.e. scaffolding, ECM• Cell signaling (i.e. immune response, cell

adhesion, cell cycles)• Antibody• Denaturation; Folding

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Biosynthesis of ProteinsBiosynthesis of Proteins

• Transcription: gene sequence mRNA• Translation: mRNA amino acids:

– Codons – same for both prokaryotes and eukaryotes, but with different frequencies

• Post translational modifications– Different between prokaryotes and eukaryotes

Central Dogma: DNA mRNA Protein

Protein StructuresProtein Structures

• Primary structure: a.a. sequence– N terminus– C terminus

• Secondary structure: local structures stabilized by hydrogen bonds– Alpha helix– Beta sheet– Random coil

Alpha HelixAlpha Helix

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Beta SheetBeta Sheet

Parallel Anti parallel

Protein Structures (Protein Structures (contcont’’dd))

• Tertiary Structure (fold): overall shape of a single protein molecule– Stabilized by nonlocal interactions– Hydrophobic core– Salt bridges– Hydrogen bonds– Disulfide bonds– Post-translation modification

Protein Structures (Protein Structures (contcont’’dd))

• Quaternary Structure: interactions of more than one protein molecules.– Protein subunits

– Protein complex

– Active sites

• Monomer, dimer, trimer, tetramer, etc.

Protein StructuresProtein StructuresN terminus

C terminus

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LIPIDLIPID

• Hydro-carbon based biomolecules that are hydrophobic (some are amphiphilic or amphiphatic)

• Water insoluble and soluble in nonpolarorganic solvents

• Consists of triglycerides• Possess a broad and diverse range of

structures• Alipathic or aromatic

CharacteristicsCharacteristics

• Ampiphilic/ampiphatic

• Alipathic/aromatic

• Cyclic/acyclic

• Branched/straight

• Flexible/rigid

DNADNA & RNA

Genome sizes vary dramatically among species. Current eukaryotic genome sizes are known to vary by more than five orders of magnitude; the genome of Amoeba dubia is roughly 200 times larger than that of humans and more than 200000 times larger than that of the microsporidium Encephalitozoon cuniculi.

The genome size or C (constant)-value of an organism is defined as the total amount of DNA containedwithin its haploid chromosome set.

ChromosomeChromosome

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• Right handed double helix• 3.4 nm per helical turn• 2 nm diameter for the helical width• 10 base pairs per turn • Two polynucleotide chains• Running in opposite directions• Held together by Hydrogen bond between base pairs

This structure was first described by James Watson and Francis Crick in 1953.

DNA: DNA: DDeoxyriboeoxyribonnucleic ucleic aacidcidDNA StructureDNA Structure

Units of DNA: nucleotides, Each nucleotide consists of:

a deoxyribose, a nitrogen containing base,a phosphate group.

Phosphodiesterbond

GC pair has 3 Hydrogen bonds AT pair has 2 hydrogen bonds

Pyrimidine ring Purine ring

RNARNA

• Messenger RNA (mRNA): bound to ribosomesand translated to protein with the help of tRNA

• Transfer RNA (tRNA): a small RNA chain of 74-95 nucleotides that transfers a specific amino acid to a growing polypeptide chain at ribosomal site

• Ribosomal RNA (rRNA): catalytic component of the ribosomes, abundant, at least 80% of the RNA molecules in a typical eukaryotic cell

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Structural differences between DNA and RNA molecules:

RNA has OH group at 2' position vs. DNA has only a hydrogen

RNA bases are A, U, G, C, while DNA bases are A, T, G, C.

•DNA lacks the 2'-OH and will not be hydrolyzed. Thus, it is a more stable polymer and better suited for storage of genetic information.

✔10. Self-replication and transcription.

✔11. Translation and reverse transcription.

✔9. Single-stranded

✔8. Two (2) chains held in a double helix by hydrogen bonds

✔✔Guanine (G) present

✔✔Cytosine (C) present

✔Uracil (U) present

✔Thymine (T) present

✔✔Adenine (A) present

7. Nitrogenous bases:

✔✔6. Polymer of nucleotides

✔✔5. It’s sugar is linked to a phosphate group at one end and a nitrogenous base at the other end

✔4. Deoxyribose sugar present

✔3. Ribose sugar present

✔2. Ribonucleic acid

✔1. Deoxyribonucleic acid

RNADNACharacteristics

• Chance to synthesize one small protein:– 1 Polypeptide with 30 amino acids– 90 base pairs in DNA sequence– Possible combination: 490 = 1.5 x 1054

(Ohio Super Lotto: 456 = 8.3 x 109 )– Probability for each mutation: 10-6

– Cell (E. coli) doubling time: 30 minutes (0.5 hrs)– Time required for evolution: (0.5 hrs)(106)(1.5x1054) = 7.5 x 1059 hrs = 8.56 x 1055 years>>> earth age ?

Creation or Evolution ?Creation or Evolution ?Creation or Evolution ?

Cost of Synthetic PeptidesCost of Synthetic PeptidesPeptide No of amino

acidsCost / gram

Amino acidAspartameBrodykininLeutinizing releasinghormone (LRH)Beta-Endorphin

129

10

31

$ 0.05$ 0.55$ 1,600$ 2,000

$20,000

From Gene (DNA) to Gene Products (Proteins)

From Gene (DNA) to From Gene (DNA) to Gene Products (Proteins)Gene Products (Proteins)

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Recombinant DNA TechnologyBioprocessing Applications

Recombinant DNA TechnologyRecombinant DNA TechnologyBioprocessing ApplicationsBioprocessing Applications

• Produce gene products (human proteins) not normally found in host cells (microorganisms)

• Construct novel biochemical pathways in cells for:– wider substrate utilization– small molecule (e.g., antibiotics) modification

• Increase gene dosage and concentration of gene products -increase metabolic rate

• Increase product yield• Put interested gene under control of known regulatory

mechanisms• Utilize gene products with special physicochemical

properties (e.g., thermostability)

Recombinant DNA TechnologyRecombinant DNA Technology

Protein SynthesisProtein Synthesisrr--DNA Technology:DNA Technology:

some website references

• http://present.smith.udel.edu/biotech/rDNA.html (This one has very good introductory movie/animation on rDNA technology that would be fun for you to watch)

• http://www.bio.miami.edu/dana/250/25003_10print.html

• http://cwx.prenhall.com/horton/medialib/media_portfolio/23.html

• http://www.biology.arizona.edu/molecular_bio/problem_sets/Recombinant_DNA_Technology/recombinant_dna.html

• http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html

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Genetic EngineeringGenetic Engineering• Mutation - point mutation: 10-6

• Sexual Hybridization - Meiosis in eukaryotes

• Parasexual Processes -– Conjugation - cell to cell contact– Transduction - phage– Transformation - plasmid

• Mitotic recombination - filamentous fungi• Protoplast Fusion• Cell Fusion - Hybridoma

Genetic EngineeringGenetic EngineeringBiosynthetic processes from gene to proteinBiosynthetic processes from gene to protein

• DNA Replication• RNA Transcription

– mRNA modification (eucaryotic cells)

• Protein Translation• Post-translational modifications

– N-terminal Methionine removal– Disulfide bond between 2 Cysteines– Pre-Pro-Protein– Glycosylation

DNA DNA

RNA

Protein

Central Dogma

Recombinant DNA TechnologyRecombinant DNA TechnologyFour Main Steps in CloningFour Main Steps in Cloning

• Obtaining DNA fragments (genetic code)

• Joining to vector• Introduction to host

cells• Selection of mutant

Recombinant DNA TechnologyRecombinant DNA TechnologyStrategies of CloningStrategies of Cloning

• How to get the foreign genetic code (DNA sequence) for the protein product?– Direct cloning– Indirect cloning - Complimentary DNA technique– PCR– Chemical DNA synthesis

♦ Human genome - 46 chromosomes– cut with EcoRI - 700,000 DNA fragments

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Synthesis of Synthesis of cDNAcDNA

The steps in the preparation of cDNA are to: (1) copy the mRNA using reverse transcriptase and a primer with dNTPs (2) digest with RNase H to nick the mRNA bound to DNA (3) add polymerase I, which will carry out nick translation, removing the RNA with its 5'-->3' exonuclease activity. (4) Finally, the double stranded DNA is cut with restriction endonucleases to generate the "sticky ends" needed to insert the DNA into a vector.

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

The sequence to be amplified is shown in blue. (1) The duplex DNA is melted by heating and cooled in the presence of a large excess of two primers (red and yellow) that flank the region of interest. (2) A heat-stable DNA polymerase catalyzes extension of these primers, copying each DNA strand. Successive cycles of heating and cooling in the presence of the primers allow the desired sequence to be repeatedly copied until, after 20 to 30 cycles, it represents most of the DNA in the reaction mixture.

Construction of Genomic DNA LibraryConstruction of Genomic DNA Library

To generate additional copies of the fragment. Before PCR, this was a key mechanism for amplifying fragments of DNA. A "restriction fragment" is a portion of the genome generated by digestion with restriction endonucleases.

Screening DNA LibraryScreening DNA Library

Colonies of cells containing recombinant molecules are grown on petri plates. A replica of the colonies is made by overlaying a filter disk on the plate. DNA and protein are released from the cells in situ and immobilized to the filter. The filter is then incubated with labeled probe under conditions in which the probe specifically recognizes the desired DNA or protein. After nonspecifically bound probe is washed away, specifically bound probe is detected by a method appropriate for the label (in this case, autoradiography). Duplicate filters are used to distinguish false positives from true positives. By aligning thefilters with the original plates, cells containing the recombinant of interest can be identified.

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Chromosome WalkingChromosome Walking

The restriction endonuclease sites (indicated by vertical arrows) are mapped on the starting recombinant. Based on this map, the terminal fragment (1) of the starting recombinant is isolated and used to probe a genomic library. The recombinants that hybridize to this fragment are restriction-mapped to identify the recombinant that extends furthest into the region of the chromosome adjacent to the first recombinant. The process is then repeated, using the restriction fragment furthest removed from the starting recombinant as the next probe.

Recombinant DNA TechnologyRecombinant DNA TechnologyHow to Join DNA Fragment to VectorHow to Join DNA Fragment to Vector

• Homopolymer tailing

• Ligation of cohesive termini produced by restriction endonucleases

• Blunt-end ligation (no linker)

• Linker molecules

Recombinant DNA TechnologyRecombinant DNA TechnologyRestriction EnzymesRestriction Enzymes

• Restriction endonucleases: e.g. EcoR1, BamH1, HindIII, etc.– sticky ends– blunt ends

• DNA ligase• DNA polymerase I• Reverse transcriptase• Terminal transferase

Restriction Fragment Length Restriction Fragment Length Polymorphisms (Polymorphisms (RFLPsRFLPs) )

RFLPs are used in many ways, such as for disease mapping, DNA fingerprinting and for examining genetic relatedness of organisms.

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Vectors (Cloning Vehicles)Vectors (Cloning Vehicles)

Yeast artificial chromosome (YAC)E. coli Plasmid pBR322

Recombinant DNA TechnologyRecombinant DNA TechnologyCloning Vehicles (Vectors) Cloning Vehicles (Vectors)

• Autonomous replication - origin of replication• Ability to accommodate foreign DNA• Easy insertion (transformation) in host

cells• Selection markers

– antibiotic resistance gene– nutrient gene for auxotroph mutant

• Contain specific target site for each of the multiple restriction endonuclease sites

Recombinant DNA TechnologyRecombinant DNA TechnologyCloning Vehicles (Vectors) Cloning Vehicles (Vectors)

• Wide range hosts - Shuttle vectors• Secretion vector plasmids• Regulation systems of expression of

cloned genes (expression vectors)• Amplification - high copy number• Be maintained stably in the host cells

Eukaryotic Shuttle Vector• Plasmids that contain a cassette of genes for

expression in eukaryotic cells as well as elements that allow plasmid replication (under the control of a bacterial origin) in bacteria and selection of plasmid-containing bacteria

• With shuttle vectors, the initial cloning steps are conducted with E. coli before the fully developed construct is introduced into a different host cell.

• Additionally, a number of vectors with a single broad-host-range origin of DNA replication are developed instead of a narrow-host-range origin, suitable for a variety of microorganisms instead of just E. coli.

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Eukaryotic Shuttle VectorEukaryotic Shuttle Vector

• Multiple cloning site for a gene of interest

• Eukaryotic selectable marker

• Origin of replication in the eukaryotic cell

• Origin of replication in bacterial cell

• Bacterial selectable marker gene

Recombinant DNA TechnologyRecombinant DNA TechnologyIntroducing Vector into Host CellIntroducing Vector into Host Cell

• Transfection with recombinant phage DNA

• Transformation with recombinant plasmid DNA

• In vitro packaging into phage coat: transduction with recombinant phage or cosmid

From:http://wilkes1.wilkes.edu/~terzaghi/BIO-226/lectures/39.html

Lambda vectorsLambda vectors

•λ phages are viruses which can infect bacteria

•Very High transformation efficiency

•Have a linear genome of ~50 kb

•Insert Size 15-20 kb

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Other Viral VectorsOther Viral Vectors

• Bacteriophage M 13 – Single stranded DNA

• Retroviruses• Adenoviruses

CosmidCosmid VectorsVectors

• Combination of plasmids and “cos” sites of λ phages • High Transformation efficiency• Insert Size can be up to ~45 kb• The vector size is ~6 kb• Uses the same packaging technique as

bacteriophage lambda• Therefore insert size can be high.• Cut open cosmid using ScaI and BamH1 • Insert foreign DNA• Only cosmids with inserts will form infective viruses

Yeast Artificial ChromosomesYeast Artificial ChromosomesEssential components• Yeast Centromeres: DNA without centromeres often get lost during

mitosis

• Telomeres: Protect ends of DNA

• Autonomous Replicating sequences: analogous to “ori” in plasmids

• Ampicillin resistance gene

• Markers like TRP1 or URA 3

• RE sites

Insert size : UP TO 2 mega basesVery Low transformation efficiencyUnstable insert (gets deleted or rearranged)

BACsBACs and PACsand PACs

• Based on the E. coli F’ plasmid: can be propagated in E. coli

• Can accommodate up to 500 kb• DNA is more stable

• Modified bacteriophage P1 to accept inserts up to 400 kb• Much more efficient than BACs at infecting hosts

Bacterial artificial chromosomes (BACs)

P1 derived artificial chromosomes (PACs)

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Methods of TransformationMethods of Transformation

• Prokaryotic cells– Ca treatment– Electroporation– Viruses– F plasmid– Conjugation

• Eukaryotic cells– Ca3(PO4)2 treatment– Electroporation– Viruses– Ballistic method

(BIOLISTICS)– DEAE dextran– Lipofection– microinjection

Recombinant DNA TechnologyRecombinant DNA TechnologyCharacterization of Cloned DNA Characterization of Cloned DNA

• Insertional inactivation (negative selection)– lacZ gene for enzyme to hydrolyze Xgal (blue colonies vs. white

colonies)

• in situ colony hybridization (P32 probe) • Plasmid DNA isolation• Southern Blotting (DNA), Northern (RNA)• Immunochemical methods (antibody probe) • DNA sequencing - up to ~1000 nucleotides

Recombinant Protein Recombinant Protein TherapeuticsTherapeutics

Recombinant protein therapeutics—success rates, market trends and values to 2010http://www.nature.com/news/2004/041206/fig_tab/nbt1204-1513_F1.html

Recombinant Protein TherapeuticsRecombinant Protein Therapeutics• Colony-stimulating factors (CSFs). Immune system growth factors that control the

differentiation, growth, and activity of white blood cells. GM-CSF stimulates the production of both granulocytes and macrophages, helping to overcome immune deficiencies and fight infection. G-CSF; M-CSF.

• Erythropoietin (EPO). A protein produced in the kidney that stimulates red blood cell production. It is used to treat anemia linked with renal failure and also find use in anemia resulting from chemotherapy or therapy for AIDS.

• Blood factors. Proteins involved in the multi-step process of blood clotting. Some, such as Factor VIII, is deficient in persons with hemophilia A.

• Human growth hormone (HGH)• Growth factors. Proteins responsible for directing the differentiation and production of

various cell types. Epidermal growth factor (EGF) for wound healing; Platelet-derived growth factor (PDGF) for collagen deposition in tissue repair; Insulin-like growth factor (IGF) for promoting tissue growth.

• Interferons. Broad-acting agents that interfere with viral infection (e.g., AIDS) and control the spread of some cancers and infectious diseases. α-, β-, γ-interferons.

• Interleukins (ILs). Immune system hormones, or cytokines, that stimulate and regulate the growth and function of a wide variety of white blood cell types, can be used in treating cancer, wound healing, immune deficiencies, and AIDS. IL-1, IL-2, IL-3.

• Monoclonal antibodies (MAbs). Widely used in biodiagnostics and treatments of infectious diseases and cancers.

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Biopharmaceutical ProductsBiopharmaceutical ProductsBiopharmaceutical Products• Market: increased from $12 billion in 2000

to over $40 billion in 2006• 122 products approved in the US and

Europe:– 50 Mammalian cells (CHO, NS0, etc.)– 39 prokaryotic cells (mainly E. coli)– 21 yeast cells (mainly Saccharomyces

cerevisiae)– 12 undisclosed systems

500 undergoing clinical evaluation – high demand for cGMPmanufacturing on small, pilot and large scales

1. Target newprotein

products

2. Justify newproperties of

existingproducts

3. Genetic engineering

cloningexpression

4. Protein engineering

5. Small-scalefermentationand purifica-

tion6. Pilot-scalefermentationand purifica-

tion

7. Product formulation

8. Toxicology studies

9. FDAacceptance

10. Commercialproduction

11. MarketingFlow chart for commercial development of recombinant protein products

Major BiologicsMajor Biologics

There are at least 23 protein therapeutics with sales of $1 billion or more.