2. GENE CLONING
Gene cloning is the process of incorporating
foreign genes into hybrid DNA replicons.
Cloned genes can be expressed in appropriate
host cells, and the phenotypes that they determine
can be analyzed.
A Clone is a population of organisms or molecules
derived by asexual reproduction from a single
ancestor.
Some key concepts underlying representative
methods are summarized here.
3. DNA CLONING
Restriction enzymes (endonucleases):
in nature, these enzymes
protect bacteria from intruding DNA; they
cut up the DNA (restriction); very specific
Restriction site:
recognition sequence for a
particular restriction enzyme
Restriction fragments:
segments of DNA cut by
restriction enzymes in a reproducable
way
Sticky end:
short extensions of
restriction fragments
DNA ligase:
enzyme that can join the
sticky ends of DNA fragments
Cloning vector:
DNA molecule that can carry
foreign DNA into a cell and replicate there
(usually bacterial plasmids)
4. STEPS FOR EUKARYOTIC GENE
CLONING
Isolation of cloning vector
(bacterial plasmid) & gene-
source DNA (gene of interest)
Insertion of gene-source DNA
into the cloning vector using the
same restriction enzyme; bind
the fragmented DNA with DNA
ligase
Introduction of cloning vector into
cells (transformation by bacterial
cells)
Cloning of cells (and foreign
genes)
Identification of cell clones
carrying the gene of interest
6. WHY CLONE IN EUKARYOTES?
Eukaryotic genes may not be expressed properly in
bacterial host
different mechanisms for gene expression
modifications (glycosylation)
very large pieces of DNA can be cloned (yACs)
Cloning in S. cerevisiae
(cloning in eukaryotes)
8. TRANSFECTION OF
EUKARYOTIC CELLS
Transfection originally referred to
transformation of viral nucleic acid into cells
(both bacterial and eukaryotic)
Microinjection- a technique where DNA can
also be injected directly into the nucleus using
micropipets
Electroporation- exposes host cells to pulsed
electrical fields in the presence of cloned DNA.
Particle gun or gene gun- high-velocity
microprojectile “gun” can be used to get DNA
into cells.
9. SHUTTLE VECTORS AND EXPRESSION
VECTORS
Shuttle Vectors
Vectors that can replicate and are stably
maintained in two (or more) unrelated host
organisms
Genes carried these vectors can be moved
between unrelated organisms. Have been
developed that replicate in both Escherichia
coli and Bacillus subtilis
Convenient marker to select for the plasmid
in yeast
Importance: DNA cloned in one organism
can be replicated in a second host without
modifying the vector
11. Expression Vectors
are designed to allow the experimenter to
control the expression of cloned
contain regulatory sequences that allow
manipulation of gene expression
Promoters from E. coli that are used in
expression vectors include lac (the lac operon
promoter), trp (the trp operon promoter), tac
and trc (synthetic hybrids of the trp and lac
promoters), and lambda PL (the leftward
lambda promoter;
13. EUKARYOTIC VECTORS
It is often desirable to clone and express
genes directly in eukaryotes, and vectors
are available for cloning into yeast
Two-micron circle- A plasmid where most
yeast vectors are based on this
Virus vectors are commonly used in
multicellular eukaryotes.
Integrating vectors
are maintained at low copy number by
integrating into the host chromosome
14. BACTERIOPHAGE LAMBDA AS A
CLONING VECTOR
Lambda can also be used as a cloning
vector for in vitro recombination
useful cloning vector because it can hold
larger amounts of DNA than most
plasmids and DNA can be efficiently
packaged into phage particles in vitro.
Bacteriophage lambda- most studied of
the specialized transducing phages
Phage lambda- has a large number of
genes.
15. MODIFIED LAMBDA PHAGES
Wild-type lambda is not suitable as a
cloning vector because its genome has too
many restriction enzyme sites
Charon phages- one set of modified
lambda phages
unwanted restriction enzyme sites have
been removed by mutation.
Replacement vectors are especially
useful in cloning large DNA fragments
16. EUKARYOTIC ARTIFICIAL CHROMOSOMES
Yeast Artificial Chromosomes (YACs)
first artificial chromosomes
replicate in yeast like normal chromosomes, but they
have sites where very large fragments of DNA can be
inserted.
To function like normal eukaryotic chromosomes
(1) an origin of DNA replication,
(2) telomeres for replicating DNA at the ends of the
chromosome and
(3) a centromere for segregation during mitosis.
(4) cloning site
(5) gene
for selection transformation
into the host
17. Human artificial chromosomes (HACs)
Have also been developed and are
similar to YACs in overall structure.
Circular*
Have long arrays of these repeats to be
inherited
Must be stably
Mammalian centromere consists of long
stretches of repeated sequences and
HACs.
18. • The choice of an expression system depends
primarily on the quality of the recombinant protein.
• However, the yield of the product and cost of
production and purification are also important
considerations.
• The vector must be designed to be maintained in
the eukaryotic host.
• The vector must have eukaryotic promoter,
transcriptional, translational stop signals, a
sequence that enables polyadenylation and a
selectable marker gene.
EUKARYOTIC EXPRESSION SYSTEMS
19. • The major features of a eukaryotic expression vector
are a promoter, a multiple cloning site, DNA segment
for termination and polyadenylation, selectable marker,
origin of replication in E. coli and eukaryotic cell and
Ampr for marker in E. coli.
20. • Saccharomyces cerevisiae
• Pichia pastoris
• Baculovirus-insect cell lines
• Mammalian systems
EUKARYOTIC EXPRESSION SYSTEMS
21. • It is the most common eukaryotic system and
there is a great deal of study about this organism.
• It is a single-celled and behaves like a bacterial
culture and can be grown in relatively simple
media in both small and large-scale production.
• Well characterized with many strong regulatable
promoters with naturally occurring plasmids.
• Carry out post-translational modifications.
• Secretes very few of its own proteins.
• Recognized as safe by USDA and FDA.
SACCHAROMYCES CEREVISIAE
22.
23. • There are three main classes of S. cerevisiae
expression vectors.
• Yeast episomal plasmids (YEps).
• Yeast integrating plasmids (YIps)
• Yeast artificial chromosomes (YACs)
• Yeast episomal plasmids have been used
extensively for the production of eitehr intra- or
extracellular heterologous proteins.
• Typically, vectors function in both E. coli and S.
cerevisiae.
SACCHAROMYCES CEREVISIAE
24. • The YEps vectors are based on the high-copy-
number 2µm plasmids.
• The vectors replicate independently via a single
origin of replication.
• There are more than 30 copies per cell.
• Selection scheme rely on mutant host strains
that require a particular amino acid (histidine,
tryptophan, or leucine) or nucleotide (uracil).
• When a Yep with a wild-type LEU2 gene is
transformed into a mutant leu2 host cell, only
cells that carry plasmid will grow.
SACCHAROMYCES CEREVISIAE
25. • Generally, tightly regulatable, inducible promoters are
preferred for producing large amounts of recombinant
protein at a specific time during large-scale growth.
26. • Most heterologous genes are provided with a
DNA coding sequence for signal peptide that
facilitates the secretion of protein through cell
membranes and external environment.
• Other sequence that protect the recombinant
protein from proteolytic degradation, and provide
a affinity tag is also used.
• These extra amino acid sequences are equipped
with a protease cleavage site so that they can be
removed from the recombinant protein.
SACCHAROMYCES CEREVISIAE
27. • Plasmid-based yeast expression systems are often
unstable under large-scale growth conditions even
in the presence of selection pressure.
• A Yip vector is used to integrate a heterologous
gene into the host genome to provide a more
reliable production system.
• The plasmid does not usually carry an origin of
replication.
• The disadvantage is the low yield of recombinant
protein from a single gene copy.
SACCHAROMYCES CEREVISIAE
29. YEAST ARTIFICIAL CHROMOSOMES
YAC is an artificially constructed chromosome that
contains a
Centromere
Telomeres
Autonomous replicating sequence (ARS) element
required for replication and preservation of YAC
in yeast cells
ARS elements are thought to act as replication origins
First described in 1983 by Murray and Szostak
30. YACs are plasmid shuttle vectors capable of
replicating and being selected in common
bacterial hosts such as Escherichia coli, as
well as in the budding yeast
Saccharomyces cerevisiae.
Yeast artificial chromosome (YAC) is
a human-engineered DNA molecule
used to clone DNA sequences in
yeast cells
31. Purpose of using YAC Vectors:
Cloning vehicles that propogate in eukaryotic cell hosts as
eukaryotic Chromosomes
Clone very large inserts of DNA: 100 kb - 10 Mb
Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule with telomeric
ends: Artificial Chromosome
32. CONSTRUCTION OF YEAST ARTIFICIAL
CHROMOSOMES
Plasmid DNA purification
Treatment with restriction
enzymes
Ligation and yeast
transformation
33.
34. • A YAC is designed to clone a large segment of DNA
(100 kb), which is then maintained as a separate
chromosome in the host yeast cell.
• It is highly stable and has been used for the physical
mapping of human genomic DNA, the analysis of
transcription units, and genomic libraries.
• It has a sequences that act as ARS for replication,
centromere for cell division, and telomere for stability.
• To date, they have not been used as expression
systems for the commercial production.
YAC cloning system
36. • Human Cu/Zn SOD cDNA was cloned between the
promoter and termination-polyadenylation sequence of
the yeast GAPD gene and subsequently used to
transform LEU- mutant host cell.
Intercellular Production in Yeast
37. • Proteins may also be produced for secretion.
• In this system, any glycosylated protein is secreted (O
or N-linked).
• The coding sequences of recombinant proteins must be
cloned downstream of a leader sequence, the yeast
mating type factor α-factor.
• Under these conditions, correct disulfide bond
formation, proteolytic removal of the leader sequence,
and appropriate posttranslational modifications occur,
and an active recombinant protein is secreted.
• The leader peptide is removed by endoprotease that
recognizes the Lys-Arg.
Secretion of Heterologous Proteins
38. • For example, a properly processed and active form of
the protein hirudin; a powerful anticoagulant protein
cloned from a leech, was synthesized and secreted by
an S. cerevisiae.
• A YEp vector that had the prepro-α-factor sequence
added to the huridin coding sequencea to allow
expression that is cleaved away in processing.
• Leaves active hirudin which is secreted.
• Producing a recombinant protein for use in human
therapeutics in yeast rather than in bacteria is to ensure
the proper folding.
Secretion of Heterologous Proteins
40. • Though S. cerevisae is successfully used to produce
recombinant proteins for human, it has major
drawbacks.
• The level of protein production is low.
• There is the tendency for hyperglycosylation resulting
in change of protein function.
• Proteins are often retained in periplasm, increasing
time and cost for purification.
• It produces ethanol at high cell densities, which is toxic
to cells.
PICHIA PASTORIS EXPRESSION SYSTEMS
41. • P. pastoris is a methylotrophic yeast that is able to
utilize methanol as a source of carbon and energy.
• Glycosylation occurs to a lesser extent and the
linkages between sugar residues are of the α-1,2 type.
• P. pastoris strain was extensively engineered with the
aim of developing a “humanized” strain that glycosylate
proteins in a manner identical to that of human cells.
• It does not produce ethanol.
• It normally secretes very few proteins, thus simplifying
the purification of secreted recombinant proteins.
PICHIA PASTORIS EXPRESSION SYSTEMS
44. Pichia pastoris vs Saccharomyces cerevisiae
Advantages P. pastoris and S. cerevisiae
Short doubling time
Readily manipulated genome
Improved folding and post-translational modification
Expression of similar genes and compatible vectors
Better yield of recombinant protein (higher cell density)
Methylotrophic yeast (methanol as its only carbon source)
Strongly methanol induced promoters
(alcohol oxidase genes: AOX1 and AOX2)
Optimal growth pH 3.0-7.0
Extremely low levels of endogenous protein secretion
Expression vectors integrated in the genome
Disulfide bond formation and glycosylation modifications
S. cerevisiae
P. pastoris
45. PRACTICAL DNA TECHNOLOGY USES
Diagnosis of disease
Human gene therapy
Pharmaceutical products
(vaccines)
Forensics
Animal husbandry
(transgenic organisms)
Genetic engineering in
plants
Ethical concerns?