1. Biofactories in the OUTLINE
Biotechnology Industry • Introduction - Biotechnology
• Biomolecules
• From Gene to Product (Protein)
From Gene to Bioproducts
• Recombinant DNA Technology
Bioprocess Engineering Workshop
Prof. S. T. Yang
Dept. Chemical & Biomolecular Eng
The Ohio State University
Biotechnology Pharmaceuticals
Industrial Markets for Biotechnology
drugs, healthcare
• Pharmaceutical industry - over $400 billion
A Diagnostics
P • Agriculture and food industry
P Human Biomedical – transgenic crops, animals
artificial organs, body parts
L – recombinant bovine somatotropin (BSA)
I Plant tissue cultures, • Chemical industry – over $2 trillion on sales worldwide
C Transgenic plants – commodity chemicals, specialty chemicals, consumer
A care products, and pharmaceuticals; plastics/polymers
Agriculture
T Transgenic animals – uses nearly $24 billion worth of hydrocarbon feedstocks
I annually in the US.
O Biochemicals • Fuel and energy
N
S Industrial – 4.5 billion gallons of ethanol from corn in 2006
Environment
pollution control – 75 million gallons of biodiesel from soybean oil in 2005
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2. Biotechnology Cell
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”
DNA account for about 0.25% cell weight for a typical mammalian cell and
-- E u r o p e a n F e d e r a t i o n o f B i o t e c h no lo gy , 1 98 5 1% for a typical bacterial cell.
Biomolecules Carbohydrates
• Carbohydrates • Contains oxygen, hydrogen and carbon atoms,
and no others. Some not
• Proteins • (CH2O)n or derivations
• Function as storage and transport form of
• Lipids energy
• Classified based on the number or sugar units:
• Nucleic acids Monosaccharide, Disaccharides,
Oligosaccharides, and Polysaccharides
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3. Protein (Polypeptides) Sizes
• Discovered in 1838 by Jons Berzelius • Total molecular mass in daltons or kDa
• From Greek, protas: of primary importance
• Yeast proteins average 466 a.a. and 53
• Consists of amino acids arranged in a
linear chain linked by peptide bonds kDa in mass.
• 20 standard amino acids • The largest known protein are the titins, a
• Plants and microorganisms can synthesize component of muscle sarcomere, MM of
all the 20 a.a., animal cannot 3,000 kDa and 27,000 a.a.
• Essential amino acids from diet
Classification Functions of Proteins
• Globular proteins • Enzymes: catalyze biochemical reactions,
– Most are soluble metabolism and biosynthesis
– Mostly enzymes, antibodies, hormones • Cell cytoskeleton, i.e. scaffolding, ECM
• Fibrous proteins
• Cell signaling (i.e. immune response, cell
– Structural
adhesion, cell cycles)
• Membrane proteins
• Antibody
– Mostly in cell membranes
– Receptors • Denaturation; Folding
– Channels
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4. Biosynthesis of Proteins
Central Dogma: DNA mRNA Protein
• 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
Protein 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 Helix
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5. Protein Structures (cont’d)
• Tertiary Structure (fold): overall shape of a
single protein molecule
– Stabilized by nonlocal interactions
– Hydrophobic core
– Salt bridges
– Hydrogen bonds
Parallel Anti parallel – Disulfide bonds
– Post-translation modification
Beta Sheet
Protein Structures (cont’d) N terminus
Protein Structures
• Quaternary Structure: interactions of more
than one protein molecules.
– Protein subunits
– Protein complex
– Active sites
• Monomer, dimer, trimer, tetramer, etc.
C terminus
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6. LIPID Characteristics
• Hydro-carbon based biomolecules that are
• Ampiphilic/ampiphatic
hydrophobic (some are amphiphilic or
amphiphatic) • Alipathic/aromatic
• Water insoluble and soluble in nonpolar
organic solvents • Cyclic/acyclic
• Consists of triglycerides • Branched/straight
• Possess a broad and diverse range of
structures • Flexible/rigid
• Alipathic or aromatic
Chromosome
The genome size or
C (constant)-value of
an organism is
defined as the total
DNA & RNA amount of DNA
contained
within its haploid
chromosome set.
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.
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7. DNA Structure
DNA: Deoxyribonucleic acid
Units of DNA: nucleotides,
Each nucleotide consists of:
a deoxyribose,
• Right handed double helix a nitrogen containing base,
a phosphate group.
• 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 Phosphodiester
bond
This structure was first described by James Watson and Francis Crick in 1953.
RNA
• Messenger RNA (mRNA): bound to ribosomes
and translated to protein with the help of tRNA
• Transfer RNA (tRNA): a small RNA chain of 74-
Pyrimidine ring Purine ring
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
GC pair has 3 Hydrogen bonds AT pair has 2 hydrogen bonds
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8. Characteristics DNA RNA
Structural differences 1. Deoxyribonucleic acid ✔
between DNA and RNA 2. Ribonucleic acid ✔
molecules: 3. Ribose sugar present ✔
4. Deoxyribose sugar present ✔
RNA has OH group at 2'
position vs. DNA has only a 5. It’s sugar is linked to a phosphate group at one end and a ✔ ✔
nitrogenous base at the other end
hydrogen
6. Polymer of nucleotides ✔ ✔
RNA bases are A, U, G,
7. Nitrogenous bases:
C, while DNA bases are A,
Adenine (A) present ✔ ✔
T, G, C.
Thymine (T) present ✔
Uracil (U) present ✔
Cytosine (C) present ✔ ✔
Guanine (G) present ✔ ✔
8. Two (2) chains held in a double helix by hydrogen bonds ✔
9. Single-stranded ✔
10. Self-replication and transcription. ✔
DNA lacks the 2'-OH and will not be hydrolyzed. Thus, it is a more stable
•
11. Translation and reverse transcription. ✔
polymer and better suited for storage of genetic information.
Creation or Evolution ? From Gene (DNA) to
Gene Products (Proteins)
• Chance to synthesize one small protein:
– 1 Polypeptide with 30 amino acids
Cost of Synthetic Peptides
– 90 base pairs in DNA sequence
– Possible combination: 490 = 1.5 x 1054 Peptide No of amino Cost / gram
(Ohio Super Lotto: 456 = 8.3 x 109 ) acids
– Probability for each mutation: 10-6 Amino acid 1 $ 0.05
– Cell (E. coli) doubling time: 30 minutes (0.5 hrs) Aspartame 2 $ 0.55
Brodykinin 9 $ 1,600
– Time required for evolution:
Leutinizing releasing 10 $ 2,000
(0.5 hrs)(106)(1.5x1054) = 7.5 x 1059 hrs = 8.56 x 1055 years
hormone (LRH)
>>> earth age ? Beta-Endorphin $20,000
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9. Recombinant DNA Technology
Bioprocessing Applications
Recombinant DNA Technology
• 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)
r-DNA Technology:
Protein Synthesis 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/Recom
binant_DNA_Technology/recombinant_dna.html
• http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/Recombin
antDNA.html
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10. Genetic Engineering Genetic Engineering
Biosynthetic processes from gene to protein
• Mutation - point mutation: 10-6
• DNA Replication
• Sexual Hybridization - Meiosis in DNA DNA
eukaryotes • RNA Transcription
• Parasexual Processes - – mRNA modification (eucaryotic cells)
RNA
– Conjugation - cell to cell contact • Protein Translation
– Transduction - phage • Post-translational modifications Protein
– Transformation - plasmid – N-terminal Methionine removal Central Dogma
• Mitotic recombination - filamentous fungi – Disulfide bond between 2 Cysteines
• Protoplast Fusion – Pre-Pro-Protein
– Glycosylation
• Cell Fusion - Hybridoma
Recombinant DNA Technology Recombinant DNA Technology
Four Main Steps in Cloning Strategies of Cloning
• How to get the foreign genetic code (DNA
• Obtaining DNA
sequence) for the protein product?
fragments (genetic
– Direct cloning
code)
– Indirect cloning - Complimentary DNA technique
• Joining to vector – PCR
• Introduction to host – Chemical DNA synthesis
cells ♦ Human genome - 46 chromosomes
• Selection of mutant – cut with EcoRI - 700,000 DNA fragments
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11. Synthesis of cDNA Polymerase Chain Reaction (PCR)
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,
The sequence to be amplified is shown in blue. (1) The duplex
removing the RNA with its 5'-->3'
DNA is melted by heating and cooled in the presence of a large
exonuclease activity. (4) Finally, the
excess of two primers (red and yellow) that flank the region of
double stranded DNA is cut with
interest. (2) A heat-stable DNA polymerase catalyzes extension
restriction endonucleases to
of these primers, copying each DNA strand. Successive cycles
generate the "sticky ends" needed to
of heating and cooling in the presence of the primers allow the
insert the DNA into a vector.
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 Library
Screening 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.
To generate additional copies of the fragment. After nonspecifically bound probe is washed away, specifically
bound probe is detected by a method appropriate for the label
Before PCR, this was a key mechanism for (in this case, autoradiography). Duplicate filters are used to
amplifying fragments of DNA. A "restriction distinguish false positives from true positives. By aligning the
fragment" is a portion of the genome generated by filters with the original plates, cells containing the recombinant
of interest can be identified.
digestion with restriction endonucleases.
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12. Chromosome Walking Recombinant DNA Technology
How to Join DNA Fragment to Vector
• Homopolymer tailing
• Ligation of cohesive termini produced by
restriction endonucleases
• Blunt-end ligation (no linker)
• Linker molecules
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 Technology Restriction Fragment Length
Restriction Enzymes Polymorphisms (RFLPs)
• Restriction endonucleases: e.g. EcoR1,
BamH1, HindIII, etc.
– sticky ends
– blunt ends
• DNA ligase
• DNA polymerase I RFLPs are used in many
• Reverse transcriptase ways, such as for disease
mapping, DNA
• Terminal transferase fingerprinting and for
examining genetic
relatedness of organisms.
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13. Vectors (Cloning Vehicles) Recombinant DNA Technology
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
E. coli Plasmid pBR322 Yeast artificial chromosome (YAC) multiple restriction endonuclease sites
Recombinant DNA Technology Eukaryotic Shuttle Vector
Cloning Vehicles (Vectors)
• Plasmids that contain a cassette of genes for
expression in eukaryotic cells as well as elements
• Wide range hosts - Shuttle vectors that allow plasmid replication (under the control of
a bacterial origin) in bacteria and selection of
• Secretion vector plasmids plasmid-containing bacteria
• Regulation systems of expression of
• With shuttle vectors, the initial cloning steps are
cloned genes (expression vectors) conducted with E. coli before the fully developed
construct is introduced into a different host cell.
• Amplification - high copy number
• Be maintained stably in the host cells • 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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14. Eukaryotic Shuttle Vector Recombinant DNA Technology
Introducing Vector into Host Cell
• Multiple cloning site for
a gene of interest • Transfection with recombinant phage DNA
• Eukaryotic selectable
marker
• Transformation with recombinant plasmid
• Origin of replication in DNA
the eukaryotic cell
• Origin of replication in
• In vitro packaging into phage coat:
bacterial cell transduction with recombinant phage or
• Bacterial selectable
marker gene
cosmid
Lambda 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
From:http://wilkes1.wilkes.edu/~terzaghi/BIO-226/lectures/39.html
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15. Other Viral Vectors Cosmid Vectors
• Combination of plasmids and “cos” sites of λ phages
• Bacteriophage M 13 • High Transformation efficiency
– Single stranded DNA • Insert Size can be up to ~45 kb
• Retroviruses • The vector size is ~6 kb
• Uses the same packaging technique as
• Adenoviruses 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 Chromosomes BACs and PACs
Essential components Bacterial artificial chromosomes (BACs)
• Yeast Centromeres: DNA without centromeres often get lost during • Based on the E. coli F’ plasmid: can be propagated in E.
mitosis coli
• Telomeres: Protect ends of DNA
• Can accommodate up to 500 kb
• Autonomous Replicating sequences: analogous to “ori” in plasmids
• DNA is more stable
• Ampicillin resistance gene
P1 derived artificial chromosomes (PACs)
• Markers like TRP1 or URA 3
• RE sites
• Modified bacteriophage P1 to accept inserts up to 400 kb
Insert size : UP TO 2 mega bases
• Much more efficient than BACs at infecting hosts
Very Low transformation efficiency
Unstable insert (gets deleted or rearranged)
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16. Methods of Transformation Recombinant DNA Technology
Characterization of Cloned DNA
• Prokaryotic cells • Eukaryotic cells
– Ca treatment – Ca3(PO4)2 treatment • Insertional inactivation (negative selection)
– lacZ gene for enzyme to hydrolyze Xgal (blue colonies vs. white
– Electroporation – Electroporation
colonies)
– Viruses – Viruses
– F plasmid – Ballistic method • in situ colony hybridization (P32 probe)
– Conjugation (BIOLISTICS) • Plasmid DNA isolation
– DEAE dextran
• Southern Blotting (DNA), Northern (RNA)
– Lipofection
– microinjection • Immunochemical methods (antibody probe)
• DNA sequencing - up to ~1000 nucleotides
Recombinant Protein Therapeutics
• Colony-stimulating factors (CSFs). Immune system growth factors that control the
Recombinant Protein 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.
Therapeutics • 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
Recombinant protein therapeutics—success rates, market trends 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.
and values to 2010
• Monoclonal antibodies (MAbs). Widely used in biodiagnostics and treatments of
http://www.nature.com/news/2004/041206/fig_tab/nbt1204-1513_F1.html infectious diseases and cancers.
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17. Biopharmaceutical Products 1. Target new 2. Justify new
properties of
7. Product
protein formulation
existing
• Market: increased from $12 billion in 2000 products products
to over $40 billion in 2006
8. Toxicology
3. Genetic
• 122 products approved in the US and engineering 4. Protein
studies
Europe: cloning
expression
engineering
– 50 Mammalian cells (CHO, NS0, etc.) 9. FDA
acceptance
– 39 prokaryotic cells (mainly E. coli) 5. Small-scale
fermentation
– 21 yeast cells (mainly Saccharomyces and purifica- 6. Pilot-scale
tion fermentation
cerevisiae) and purifica- 10. Commercial
tion production
– 12 undisclosed systems
Flow chart for commercial development
500 undergoing clinical evaluation – high demand for cGMP of recombinant protein products 11. Marketing
manufacturing on small, pilot and large scales
Major Biologics
There are at least 23 protein therapeutics with sales of $1 billion or more.
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