PR3104 PHARMACEUTICAL BIOTECHNOLOGY

AY 2013/14
Dr Chew Eng Hui (Module Coordinator)
Office: S4, #03-05
Phone: 6516 1955
Email: phaceh@nus.edu.sg
Dr Rachel Ee
Office: S4, #03-04
Phone: 6516 2653
Email: phaeplr@nus.edu.sg
A/P Victor Yu
Office: S4, #03-07
Phone: 6516 8216
Email:
phayuv@nus.edu.sg
Scope of Study (RE)
Basic molecular biotechnology
Tools & techniques used when working with DNA.
Tools & techniques needed to clone and identify genes: host cells, vectors, restriction
enzymes etc, Polymerase Chain Reaction, site-directed mutagenesis.
Physicochemical properties of therapeutic proteins
Protein structure: amino acids, peptide bond, ionization, intermolecular forces, protein
folding, protein stability, solubility, hydrophobicity.
Production of therapeutic proteins
Downstream processing: isolation of proteins from cells, purification and identification.
Formulation of therapeutic proteins into dosage forms.
Insulin as a recombinant protein
Reference Materials
Gene cloning and DNA analysis: an introduction / T.A. Brown QH442.2
Bro 2006 RBR or QH442.2 Bro 2010
Pharmaceutical biotechnology: fundamentals and applications / edited
by Daan J.A. Crommelin, Robert D. Sindelar, Bernd Meibohm RS380
Pha 2008 RBR
Biotechnology and Biopharmaceuticals Transforming Proteins and
Genes into Drugs / Rodney J. Ho and Milo Gibaldi Second edition;
Full-text online via NUS Libraries
Assessments
1 online MCQ self-assessment/ refresher on Basics of DNA
structure and function/ Posted on IVLE on Jan 17 / Due Jan 30
CA1 MCQ and short structured questions / L1-7 / Mar 3
Tutorial and CA Review / L1-7 / Mar 6
Definition of Biotechnology
Using living things to create products or to do tasks for
human beings
Biotechnology was termed in 1919 by Karl Ereky - converting
raw materials into a more socially useful product
Used years ago to produce foods and increase crop yields
Traditional Biotechnology
Modern biotechnology
Recombinant DNA techniques (rDNA or genetic engineering)
PreRecombinantDNAEra
(Before1970s)
PostRecombinantDNAEra
Translation of biological molecules into therapeutic products
1940s
1970s
1980s
1990s
2000s
Taken from Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes into Drugs, 2
nd
Edition, Rodney J.Y. Ho.
PreRecombinantDNAEra
(Before1970s)
PostRecombinantDNAEra
The discovery of protein, cell, bacteria and Mendelian genetics in 1830-1900, and the
innovative milestones in modern genetics and molecular engineering, provided the basis for
exponential growth in the ability to identify, validate and produce biological molecules for
therapeutic applications.
NOVO STORY OF INSULIN
HTTP://WWW.YOUTUBE.COM/WATCH?V=CZEPQ3KKWHO
An example of biopharmaceutical production in the pre-
recombinant-DNA Era
Emergence of Biopharmaceuticals
Identification of biomolecules (e.g. antibodies, blood products,
insulin)
Widespread use is possible if sufficient quantities are collected
Produced naturally in exceedingly low amounts
Chemical synthesis/semi-synthesis not useful for large proteins
Two discoveries (mid-1970s) overcame such difficulties
Emergence of Biopharmaceuticals
1. Genetic Engineering
Facilitates the large-scale production of virtually any protein once its amino acid
sequence has been determined.
Discovery highlights:
Restriction enzymes
DNA Polymerases (for sequencing and amplifying DNA)
Manipulating (cloning and mutagenesis) and propagating DNA using
bacterial plasmids
PCR Technology
2. Hybridoma Technology
Facilitates the large-scale production of
monoclonal
antibody
Investment by biopharmaceutical companies
Pioneering
biotech cos.?
Genentech
Chiron
Immunex
Cetus
Taken from Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes into Drugs, 2
nd
Edition, Rodney J.Y. Ho.
Worldwide Market for Biopharmaceuticals
Global pharmaceutical sales in 2010 - $850 bln
Biotech drugs/biologics accounted for $140 bln (2010)
~12% (2001); 19% (2006); 29% (2011) of worlds pharmaceutical market
Protein drugs
$33 blns in 2004
Annual growth rate of 12% from 2003 through 2008
Affected by financial crisis in 2008, but showing signs of rebound in 2010.
Source: IMS Health, Nature Biotechnology 29, 585-591 (2011)
Growth trends in US biotech
market for biologics (2007-2011)
Nature Biotechnology 30, 1191-1197 (2012)
Top 25 biotech drugs based on worldwide sales
Taken from
Biotechnology and
Biopharmaceutical
s: Transforming
Proteins and
Genes into Drugs,
2
nd
Edition, Rodney
J.Y. Ho.
Pharmaceutical Biotechnology- Focus on Health & Medicine
Use of living things (e.g.bacteria) to create pharmaceutical products
Ronald A. Rader. Nature Biotechnology 26, 743 - 751 (2008)
Biotech Products
Larger M.W.
Derived from living sources human
& animal tissues, cells &
microorganisms
Not easily characterized and refined
to high degree of purity.
Often called by the same name
despite modifications in one or more
amino acid residues.
Insulin-human
Insulin-beef
Insulin-pork
Insulin-aspart etc
Key differences between biotech and chemical products
Traditional Drugs
M.W. typically < 1000Da
Can be chemically synthesized
and purified to homogeneity
Chemical modification usually
leads to drastic changes in
activity and new drugs for new
uses.
Why Biopharmaceuticals?
Proteins are the basic building blocks of life that control diverse cellular and
physiological processes such as metabolism (energy expenditure), the immune
response (involved in fighting diseases) and memory and learning in the human
brain. They play a critical role in the processes and interactions involved in
maintaining good health in the face of disease and ageing.
Therefore, protein-based drugs (biopharmaceuticals) may be a more natural
way of treating diseases compared to artificial small molecules.
Behave more
predictably
Fewer SE
Majority (>95%) of biopharmaceuticals are protein products.
Proteins are susceptible to protease degradation and
denaturation in biological fluids.
Dosage forms that can be administered by IV, IM or SC routes
are available.
Distribution of proteins (macromolecules) to tissues is limited by
the permeability (porosity) of vasculatures
Absorption and Disposition?
Immunogenicity?
Challenges of using Biopharmaceuticals
1797: Jenner inoculates child with viral vaccine to protect him from smallpox
1857: Pasteur proposes that microbes cause fermentation.
1928: Penicillin is discovered by Flemmng.
1944: Avery demonstrates that DNA is the "transforming factor" and material of genes.
1953: Double helix structure of DNA is first described by Watson and Crick.
1973: Cohen and Boyer develop genetic engineering techniques to "cut and paste" DNA and reproduce the new DNA in
bacteria.
1977: Genentech scientists and their collaborators produce the first human protein (somatostatin) in a bacterium (E. coli).
1978: Genentech scientists and their collaborators produce recombinant human insulin.
1979: Genentech scientists produce recombinant human growth hormone.
1981: First transgenic animal.
1982: Eli Lilly and Company markets Genentech-licensed recombinant human insulin - the first such product on the market.
1983: Polymerase chain reaction (PCR) technique conceived (will become a major means of copying genes and gene
fragments).
1986: Genentech receives FDA approval for Protropin for growth hormone deficiency in children - the first biotech drug
manufactured and marketed by a biotech company.
1990: Human Genome Project (HGP), an international effort to map all the genes in the human body, is launched.
1994: BRCA1, the first breast cancer susceptibility gene, is discovered.
1995: The first full gene sequence of a living organism other than a virus, is completed for the bacterium Haemophilus
influenzae.
2000: First draft of human genome sequence completed by the HGP and Celera Genomics.
Significant scientific milestones in biotechnology
http://www.accessexcellence.org/RC/AB/BC/
Capturing the History of Biotechnology
<http://www.lifesciencesfoundation.org/index.html>
Recombinant DNA Technology
Genetic Engineering
Gene Cloning
Media on Genetic Modification :
http://www.pbs.org/wgbh/nova/genome/media/2809_q056_15.html
1. Isolation of gene of interest cut & paste
2. Introduction of gene to expression vector
3. Transformation into host cells
4. Selection of the required sequence and
propagation of cells
5. Isolation & purification of protein
6. Formulation of protein product
Synthesis of a recombinant protein:
Six-Step Process
Gene cloning
21
Overview of Gene Cloning
Ability to recombine
segments of DNA from
diverse sources into
new composite
molecules, or
recombinants.
2. Host cells
1. Vectors
3. Transformation &
transfection
4. Selection of
required rDNA
22
What is PCR?
Polymerase Chain Reaction invented by Kary Mullis (1985)
Produce millions of copies of a specific DNA sequence in a short time
(approx 2h). This automated process bypasses the need to use bacteria for
amplifying DNA.
Use of a thermocycler.
23
Equipment that enables a mixture of
DNA and reagents to be incubated at a
series of temperatures that are varied in
a preprogrammed manner.
Both gene cloning and PCR can provide pure samples of an
individual gene, separated from other genes.
Cloning DNA in a test-tube
Cell-free DNA Cloning
PCR Methodology
Ingredients:
DNA, Taq DNA polymerase, Specific primers,
nucleotides dNTPs (dATP, dCTP, dTTP, dGTP),
Mg
2+
Steps:
1. Mixture is heated to 94C (1 min).
2. Mixture is cooled down to 50C-60C to allow
annealing of primers (1 min).
3. Temperature raised to 74C to allow Taq DNA
polymerase to catalyze addition of nucleotides (1.5
min)
4. The cycle of denaturation, annealing and synthesis
is repeated. After Cycle 30, > 1 billion identical
copies (2
30
= 1.07 x10
9
).
denaturation
annealing
synthesis
24
Principle of PCR in animation:
http://www.youtube.com/watch?v=2KoLnIwoZKU
CSI PCR in 60s
http://www.youtube.com/watch?v=6iFDphWXjw4
PCR Methodology
1. Sequences of primer annealing sites must be known.
Easy to synthesize a primer with a predetermined sequence, but if the sequences of
the annealing sites are unknown, appropriate primers cannot be made.
2. Length of DNA sequence that can be copied by PCR.
5 kb can be copied fairly easily. Segments up to 40kb have to be dealt with by using
specialized techniques.
3. Infidelity of DNA replication
Taq DNA polymerase has no 3 5 exonuclease (proofreading function). Error due
to base misincorporation during DNA replication.
High frequency: 1 kb sequence -> 20 cycles -> ~40% of the new DNA strands will
contain an incorrect nucleotide.
Overcome by using alternative heat-stable DNA polymerases with 3 5
exonuclease activity. E.g. Pyrococcus furiosus (Pfu) DNA pol. Reduced error rate
3.5%
26
Limitations of PCR
Other applications:
Amplify DNA fragment for gene cloning
Diagnostic applications:
1. PCR amplification of mutant alleles to determine if person is carrier
of a genetic disease (sickle cell anaemia)
2. Early detection of disease to prevent onset, e.g. PCR amplification of
DNA of a disease-causing virus.
Forensic identification (DNA profiling)
Archaeology (sex identification)
etc
etc
etc
27
Other Applications of PCR
Other Tools and Techniques for DNA work
DNA purification from cells
Gel electrophoresis
Restriction enzymes
DNA sequencing
28
Basic steps Preparing total cell DNA
Cell lysis
Lysozyme digests polymeric component of cell wal
EDTA (ethylenediamine tetraacetate) removes Mg ions essential for preserving
structure of cell membrane and inhibits enzymes that could degrade DNA.
SDS (sodium dodecyl sulfate) Detergent; removes lipids and disrupts membranes.
Preparation of cell extract
DNA purification from cells
Sources of total cell DNA to isolate and purify:
Bacteria, plants or animals
Common Methods:
1. Contaminants removal by organic extraction:
Phenol or phenol/chloroform mixture precipitate proteins,
leave nucleic acids in aqueous solutions.
31
2. Ion-exchange chromatography
Principle:
- DNA, RNA and some proteins
(in decreasing order) are
negatively charged bind to
positively charged resin.
- Electrical attachment disrupted
by salt DNA can be removed
from resin and collected.
32
DNA purification from cells
Concentration of DNA by ethanol precipitation (in the presence of salt
monovalent cation e.g. Na+ and low temperature of < -20C):
Ethanol mixed with a dilute DNA solution DNA precipitates
and can be collected by centrifugation.
Measurement of DNA concentration:
By UV spectrophotometry; absorbance measured at 260 nm.
Abs (A
260
) of 1.0 = 50 g of double-stranded DNA per ml.
Purity of DNA sample: ratio of absorbance at 260 and 280 nm
(A
260
/A
280
) should be 1.8. If < 1.8, sample contaminated
with protein or phenol.
Concentration and measurement of DNA samples
33
Gel Electrophoresis
(a) Standard electrophoresis does
not separate DNA fragments of
different sizes, whereas (b) gel
electrophoresis does.
Gel electrophoresis
Principle:
Electric current used to separate different-sized molecules in a
porous, sponge-like matrix. Smaller molecules move more
easily through the pores than larger-sized molecules.
DNA characteristics:
Highly negatively charged due to phosphate groups in the
backbone; migrate towards the positive electrode.
DNA molecules of the same length will move through the gel at
the same rate.
35
Gel submerged in salt solution that
conducts electricity
1.
Tracking solution added
to colorless DNA solution to
visually track DNA
migration through the gel
2.
Bands visualized by using
the ethidium bromide dye
or radioactivity
(autoradiography).
3.
anode
36
Gel characteristics:
Agarose gel (made from
highly purified seaweed) or
polyacrylamide (PA) used to
separate DNA molecules from
several to 50,000 nucleotides
in length.
Size resolution optimized by
gel concentration
Separation characteristics for agarose and
polyacrylamide gels
Gel type Separation range (bp)
0.3% agarose
0.7% agarose
1.4% agarose
4% PA
10% PA
20% PA
50,000 to 1,000
20,000 to 300
6,000 to 300
1,000 to 100
500 to 25
50 to 1
Gel electrophoresis
37
Estimation of Sizes of DNA molecules
38
(a) A rough estimate of fragment size can be
obtained by eye.
(a) A more accurate measurement is gained by
using the mobility of the HindIII fragments to
construct a calibration curve; the sizes of
unknown fragment can be determined from
the distances they have migrated
Restriction Endonucleases
Gene cloning requires that DNA molecules be cut in a precise and
reproducible fashion.
39
Reasons for cutting DNA:
Single gene to be cloned may consist
only 2-3kb; to be cut out of the large
(>80kb) DNA molecules.
Large DNA to be broken down to produce
fragments small enough to be carried by
the vector.
Reasons for cutting vector:
Open up the circle so that new DNA can
be inserted.
To be cut at exactly same position on the
circle.
Bacterial enzymes that recognize specific 4- to 8-bp sequences, called
restriction sites, and then cleave both DNA strands at this site.
Type I, II and III. Type II are the cutting enzymes impt in gene cloning.
Three types of restriction fragments:
Blunt ends
Protruding (sticky) 3 ends
Protruding (sticky) 5 ends
Sticky ends (also called cohesive ends) are complementary to and base pair
with other fragments generated by the same restriction enzyme.
Fragments can be covalently ligated by action of DNA ligase.
Restriction Endonucleases
Restriction Endonucleases
Note:
Restriction enzymes with different recognition sites may produce same sticky ends.
e.g. BamHI (GGATCC) and BglII (AGATCT) GATC sticky ends
Sau3A (GATC)
Performing a restriction digest in the lab
DNA sequencing
Objective: To determine sequences of bases in DNA.
Steps involved:
1. Preparation of DNA fragments
Generate a set of overlapping fragments that terminate at
different bases and differ in length by 1 nucleotide. (Nested
fragments)
2. DNA sequencing
Maxam-Gilbert, Sanger-Coulson
3. Detection step
Gel electrophoresis, Autoradiograph, Capillary electrophoresis
using fluorescent labels
43
Maxam-Gilbert (chemical) sequencing
Uses chemicals to cleave DNA at specific bases, resulting in
fragments of different lengths (chemical degradation of DNA).
Advantages:
- Requires double-stranded DNA fragments,
so need not be cloned in a plasmid vector.
- No primers needed.
- Direct sequencing of small fragments possible.
Disadvantage:
Not suitable for large scale use (difficulty to be automated);
Chemicals used are toxic (pose health hazard).
44
G
G
1. Double-stranded fragment labelled at 5 end with
32
P.
2. Labelled DNA sample denatured by heating
(90 C). Results in breakdown of base
pairing and dissociation into two components
strands.
3. Strands were separated from one another by
gel electrophoresis. One strand purified from
gel and divided into 4 samples, each of which
is treated with one of the cleavage reagents.
45
Maxam-Gilbert (chemical) sequencing
Nucleotide Cleavage agent
G alone DMS, piperidine
A+G DMS, formic acid and piperidine
C+T Hydrazine, piperidine
C alone Hydrazine in high salt
G A+G C+T C
5. Bands not duplicated in A+G lane read
as A. Bands not duplicated in C+T
read as T.
6. Sequence read from bottom of gel
up (5 to 3).
46
4. Reactions are controlled so that each
labelled strain is likely to be broken once
only.
Parallel gel electrophoresis
and autoradiography
Sanger-Coulson (chain termination) sequencing
Requires single-stranded DNA
Cloned into special vectors e.g. M13 vector or phagemids
Double-stranded DNA converted to single stranded by
denaturation with alkali or boiling
Thermal cycle sequencing using one primer
Involves enzymatic DNA polymerase synthesis of a second
strand of DNA, complementary to existing template.
Chain terminates with the use of dideoxynucleotides.
48
Sanger-Coulson (chain termination) sequencing
49
Position where OH of dNTP is replaced
by H;
Phosphate group cannot be added to
elongate the chain
G
C
C
A
A
A
C
C
G
3'
5'
1. Incubation with DNA polymerase, primer, dATP,
dCTP, dGTP, dTTP and dideoxyGTP
primer
3'
G
C
C
G
C
A
A
A
C
C
G
5'
2. Short primer
initiates
replication
process
3'
G
C
C
G
C
G
A
T
A
T
A
T
C
G
C
G
5'
3'
G
C
C
G
C
G
A
A
A
C
C
G
5'
3. DNA polymerase catalyses
formation of complementary
strand
4. DNA biosynthesis
stops when dideoxy
base added.
3'
G
C
C
G
C
G
A
T
A
T
A
T
C
G
C
G
G
5'
50
The parent sequence
C T A G
C
G
G
T
T
T
G
A
G
G
C
C
A
A
A
C
T
C
Sequenced DNA
Parent sequence
The autoradiogram provides
the sequence of the
replicated single-stranded
DNA of the parent
sequence
5
3
5
3
5. This process is repeated separately with the other 3 dideoxy bases (4 concurrent strand synthesis
reactions). Thus, 4 separate reactions result in 4 families of terminated strands.
6. The double stranded DNA can be separated by heating, and the fragments are separated by
electrophoresis.
51
Fluorescent probes
are used for
automated sequencing
(different fluorescent
labels attached to
each type of
dideoxynucleotide)
http://www.dnalc.org/ddnalc/resources/cycseq.html
52
Automated DNA sequencing
http://www.youtube.com/watch?v=SRWvn1
mUNMA
1. Isolation of gene of interest cut & paste
2. Introduction of gene to expression vector
3. Transformation into host cells
4. Selection of the required sequence and
propagation of cells
5. Isolation & purification of protein
6. Formulation of protein product
Synthesis of a recombinant protein:
Six-Step Process
Gene cloning
53
Basic features:
1. Able to replicate within host cell.
2. Contains a site where DNA can be inserted (restriction sites).
3. Contains selective marker(s) e.g. antibiotic resistance
4. Relatively small, < 10 kb in size.
Examples:
Plasmids, bacteriophage, cosmids, bacterial and yeast artificial
chromosomes (BAC/YAC), etc.
Vector for gene cloning (Cloning vector)
54
A DNA molecule that serves as a vehicle to transport a gene into host
cells.
5-10kb 12-20kb 35-45kb ~300kb ~1000kb
Plasmid vectors for use in E. coli
55
Circular molecules of DNA that lead an independent existence in a host cell.
Found naturally in bacteria and some yeasts.
Carry one or more genes responsible for useful characteristics displayed by
the host bacterium e.g. antibiotic resistance gene (selectable marker).
Generally dispensable (not essential for cell growth and division).
Possess at least one DNA sequence that acts as origin of replication
multiply independently of bacterial chromosome.
Or replicate by inserting themselves into the bacterial chromosome
(episome).
Non-integrative plasmid
Episome
56
Replication strategies for plasmids
pBR322 prototype vector
used with E. coli.
Characteristics:
Small size
2 antibiotic resistance genes
Variety of restriction sites
High copy no.
Naturally occurring plasmids extensively modified to produce vectors with
desired characteristics in genetic cloning.
Plasmid vectors for use in E. coli
57
Plasmid vectors for use in E. coli
58
pUC8 a lac selection plasmid
Advantages over pBR322:
Higher copy no.
Identification of recombinants a single step process
Plating on agar containing ampicillin and X-gal
Clustering of restriction sites
Insertional inactivation of Tet
R
:
Transformed cells plated onto Amp
agar, and replica plated onto Tet agar.
Colonies that grow on Tet agar are
Amp
R
Tet
R
non-recombinants.
Colonies that carry inserted DNA do
not grow on Tet agar (Amp
R
Tet
S
)
position on amp agar plate now known.
59
Selection of recombinant clones
1. Insertional inactivation of an antibiotic resistance gene:
Amp
R
gene has restrictions sites PstI, PvuI and ScaI. Tet
R
gene has BamHI and SaII restriction sites.
Insertion of new DNA in one of these sites inactivates gene.
e.g. pBR322
2. Insertional inactivation of lacZ gene (Lac selection)
Unmodified lacZ gene codes for the galactosidase (gal) enzyme (breaks
down lactose to glucose and galactose)
Lac promoter (strong) is induced by the addition of IPTG [isopropyl-
thiogalactoside]
Switches on gene transcription of lacZ gene to produce the gal enzyme
LacZ gene is modified from lacZ gene
Codes for part of the gal enzyme (-peptide portion); Used in mutant
E.coli strains (E. coli lacZ
-
) that have a modified lacZ gene that lacks the
segment of gene referred to as lacZ gene.
60
Selection of recombinant clones
e.g. pUC8
In the presence of IPTG,
61
Selection of recombinant clones
Using lacZ gene
Foreign gene is inserted in the
plasmid in a way that disrupts the
lacZ gene.
62
Selection of recombinant clones
With the insertion of the foreign gene, the lacZ gene is disrupted and results in
unsuccessful production of the gal enzyme component.
Lac selection
63
X-gal: lactose analog broken down by
-gal to blue colour pdt
Selection of recombinant clones
Bacteriophage vectors for use in E. coli
Viruses that infect bacteria.
Consist DNA or RNA genome and a capsid (protein coat).
Used to carry DNA fragments too large to be handled by
plasmids.
M13 and phage commonly used as cloning vectors.
Used to make DNA libraries.
64
phage M13 phage
Phages classified as temperate or virulent, depending on their life cycles.
Lytic:
Enters bacteria,
produces more
phages and kill
bacterial cells.
Lysogenic:
Integrate into
chromosome,
remains quiescent
without killing cells.
Virulent phages exhibit lytic life cycle only.
Temperate phages exhibit lysogenic life cycle, but may undergo lytic
response when conditions are suitable. Eg: Lambda () phage
65
Bacteriophage vectors for use in E. coli
Lysogenic infection cycle of bacteriophage
66
Episomal insertion
Triggering agents:
UV, DNA damaging
agents
phage genome (49 kb) impt features as a cloning vector
Must be capable of lytic growth, other viral functions irrelevant.
Genes involved in lysogenic pathway and other viral genes not essential for
lytic pathway removed Deleted genome non-lysogenic, can follow only
lytic infection cycle. Desirable feature for cloning vector (induction not
needed before plaques are formed).
Replaced with DNA (12 20 kb) to be cloned.
Foreign gene can be
inserted
67
Bacteriophage vectors for use in E. coli
68
2 conformations of DNA molecule: linear and circular forms.
Linear form: consists of two complementary strands of DNA with
short single-stranded 12-nucleotide stretch at two free ends.
Sticky or cohesive ends called cos sites
Bacteriophage vectors for use in E. coli
69
cos sites
1.Allows linear DNA molecule
injected into host cell to circularize.
2.Rolling circle mechanism of
replication results in a catenane
consisting of a series of linear
genomes joined together at the cos
sites.
Cos sites recognized by
endonucleases to cleave the
catenane to produce individual
genomes.
Bacteriophage vectors for use in E. coli
http://www.youtube.com/watch?v=ehbZpo8oXSs
Use it like a plasmid vector cloning, but not efficient.
Modifications required for greater number of recombinants.
70
Bacteriophage vectors for use in E. coli
Recombinant DNA
In vitro phage assembly
+ protein for
packaging
DNA)

Use as recombinant phage

71
Bacteriophage vectors for use in E. coli
E. coli cells infected
with mutant a
protein required for
packaging DNA
into preassembled
phage heads
missing.
Cells accumulate
empty heads.
Other Cloning Vectors
Cosmid
Hybrid between plasmid and vector.
Size of insert: 40-45 kb.
Bacterial Artificial Chromosome (BAC)
Based on the F. plasmid from E. coli
which is much larger than the standard
plasmid vectors.
Size of insert: 300 kb.
Yeast Artificial Vector (YAC)
Vector containing yeast genes.
Size of insert: 1 Mb.
72
1. Isolation of gene of interest cut & paste
2. Introduction of gene to expression vector
3. Transformation into host cells
4. Selection of the required sequence and
propagation of cells
5. Isolation & purification of protein
6. Formulation of protein product
Synthesis of a recombinant protein:
Six-Step Process
Gene cloning
73
Insufficient folding
of complex
proteins of higher
organisms
inclusion bodies
Lack of post-
translational
modifications
Endotoxins
Host Cells
Insect &
Mammalian
Bacteria Yeast Transgenic Plants
& Animals
Post-translational
modifications
differs from
mammalian cells
Problematic cell
disruption
Protease that
degrade foreign
proteins
Laborious
construction of
over-expressing
strains
Expensive media
Low growth rates
Difficult scale-up
Long
developmental
cycles
Contamination
problems
Animal viruses
prions
74
e.g. E.coli
CHO cells
Pharming
Production of Recombinant Protein in E.coli
Source of gene for cloning
Source material: Nucleic acid molecules in the form of mRNA or genomic DNA
mRNA Genomic DNA
Represents the coding sequence of a gene,
with any introns removed during RNA
processing Production of a recombinant
protein > straightforward.
Represents the genetic information that is
being expressed by the particular cell type
from which it is prepared.
If gene of interest is highly expressed, mRNA
will be in abundance, making isolation of
clones easier.
Contains non-coding DNA such as
introns, control regions and repetitive
sequences.
Represents the full complement of DNA
contained in the genome of a cell or
organism.
For studies on control of gene
expression, isolation of control
sequences is necessary, genomic DNA
is the only alternative.
76
Cloning from mRNA: cDNA synthesis
It is not possible to clone mRNA directly, so it has to be converted into cDNA
(complementary DNA) before being inserted into a suitable vector.
mRNA
Oligo(dT) primer binds to
poly(A) tract at 3end
RTase synthesizes a copy
of mRNA to produce a
cDNA-mRNA hybrid
mRNA breakdown with alkali or
RNaseH
Duplication by DNA polymerase
What primers to use?
Double stranded
cDNA
cDNA http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
RNA-priming
using oligo-dT
ss cDNA
(1
st
strand cDNA)
77
How to obtain
mRNA from cells?