BOCM 3714

Lecture 5

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 Cloning vs Expression vectors
Features Cloning vector Expression vector
Definition A small, circular DNA used Plasmid that not only introduced gene
to introduce foreign gene of interest, facilitate expression of the
of interest into host cell gene to produce protein.

Role To obtain multiple copies Used to analyse gene product: RNA or

of foreign gene of interest
Types Plasmids, cosmids,
phages, BACs, YACs,
MACs
Features Origin of replication,
multiple cloning site or
polylinker (unique
restriction sites), antibiotic
resistance gene
protein
Plasmids
Features of cloning vector, regulatory
elements (promoters, termination
sequences, transcription initiation,
translation initiation)
 Production of proteins

• Production of proteins – important application of

recombinant DNA technology

• Expression of bacterial proteins:

– Cloning of structural gene
– Expression vector: transcriptional & translational control sequences
– Use relaxed control plasmid (high copy number) and efficient
promoter – produce high amount of recombinant protein
– Overproducer

• Encounter problems
 Production of proteins

1. Large amounts of protein, useless to bacterium – insoluble

and denatured proteins in inclusion bodies
– Denature misfolded proteins by dissolving in urea or guanidinium ion,
followed by renaturation of proteins by the slow removal of solvents
through e.g. dialysis
– Can add a bacterial signal peptide – secrete protein into the
periplasmic space
– Signal peptide removed with bacterial protease
2. Protein maybe toxic for host cell, killing bacteria before
sufficient amounts of protein is produced
– Circumvent problem by placing gene encoding toxic protein under
control of an inducible promoter
– lac promoter
• Binding of lac repressor –
prevent expression of
foreign protein

• Cells grown to high

concentration – High cell
mass

• Induced added - release

repressor

• Produce large amounts of

foreign protein

• Isopropylthiogalactoside
(IPTG) analog of
allolactose
3. DNA digested with 1
restriction enzyme can be
ligated with cloning
vector cut with same
enzyme in any direction
– 50% in wrong direction in
relation to transcriptional
and translational sequences
– Protein not properly
expressed
– Use directional cloning
– Make use of 2 restriction
enzymes
 Production of Eukaryotic proteins in
bacteria
• Production of eukaryotic proteins in bacteria – several
problems
1. Eukaryotic control elements for RNA and protein synthesis
not recognised by bacterial hosts
2. Bacteria cannot carry out gene splicing
3. Bacteria lack enzymes to carry out post-translational
processing
4. Eukaryotic proteins maybe degraded by bacterial
proteases
Problems encountered with expression
of eukaryotic proteins in bacteria
• Eukaryotic control elements for RNA and protein synthesis
not recognised by bacterial hosts
– Insert protein encoding part of eukaryotic gene into vector containing
bacterial control elements
• Bacteria cannot carry out gene splicing
– Clone cDNA derived from mature mRNA
– Many mRNAs – not abundant
– If sequence known: chemically synthesize
– Eukaryotic genes: large, not stably synthesized
Problems encountered with expression
of eukaryotic proteins in bacteria
• Bacteria lack enzymes to carry out post-translational
processing
– Currently no general approach to address this problem
• Development of cloning vectors that can propagate in
eukaryotic hosts such as yeast or cultured cells, overcome
these problems
– Baculovirus-based vectors that replicate in cultured insect cells
– Also mammalian expression
– Shuttle vectors: can propagate in yeast and bacteria
• Eukaryotic proteins maybe degraded by bacterial proteases
– Can express eukaryotic protein fused to bacterial protein
– Fused at N-terminal: protected from degradation
– Remove with specific protease
– Can aid in purification process
 Application of recombinant
DNA technology
• The ability to produce proteins in large quantities –
enormous impact in medical, agricultural & industrial fields
– Human insulin: diabetes
– Human growth hormone: treat children of abnormal short stature
– Hepatitis B vaccine
– Blood clotting factors: hemophilia
 Site-directed mutagenesis

• Can manipulate proteins by

altering bases coding for
amino acids
• Oligo containing altered
bases used as primer
• Primer extended with DNA pol
I, generate mutated gene
• PCR often used
• Subtilisin – used in laundry
detergents containing bleach
• Wild type inactivated by
bleach
Reporter Genes
• A reporter gene is a known gene
whose RNA or protein levels can be
measured easily and accurately.

• Often used to replace other coding

regions whose protein products are
difficult to measure quantitatively.
Green fluorescent protein (GFP) – bioluminescent
jellyfish
• Fluoresces green when irradiated by
UV or blue light
• Fluorescence result of spontaneous
cyclization and oxidation by O2 of Ser-
Tyr-Gly
• Double bonds
• Require no substrate or co-factor

Biotek Instruments
• Presence monitored through UV or blue light
• Can be fused to another protein – monitor expression
• GFP variants – different excitation and emission
wavelengths
• Allows for simultaneous detection
 Purification and detection tags:
fusion proteins

• Reporter genes can be attached to

other sequences so that the reporter
protein is synthesized fused to
another protein.

• Often a short peptide sequence that

serves as an affinity or epitope tag
(antigenic determinant) is used.
Transgenic organisms & Gene Therapy

• Manipulate an intact organism, not only protein

• Multicellular organism expressing foreign gene –

transgenic

• Change to be permanent – transgene must be

stably integrated into organism’s germ cells
• Microinjection DNA
containing the rat
growth hormone into
nuclei of fertilized
mouse eggs

• “supermice”

• Genetically altered
animals - transgenic
 Cloning of transgenic animals

• Cloned herds of
transgenic or gene-
targeted farm animals
that produce valuable
human proteins.

• Produce human
growth hormone,
blood clotting factors
in milk – “pharm
animals”
 Transgenic plants
1 • Crop plants engineered for
increased herbicide
resistance
2 • Altered plant architecture
(height) for improved
productivity
3 • Improved tolerance to
environmental stresses,
drought
4
• Improved nutritional
properties
– Rice contain genes encoding β-
carotene
– Precursor for vitamin A
– Vitamin A deficiency affects
millions, causing death &
blindness
 Gene Therapy

• Transfer of new genetic material to cells of an individual

resulting in a therapeutic benefit

• Three types:
1. Ex vivo: cells removed from body, incubated with vector and return –
bone marrow cells
2. In situ: vector applied directly to affected tissues – used in treatment
of cystic fibrosis by inhaling an aerosol containing a vector encoding
the normal protein
3. In vivo: vector injected directly into bloodstream
 Summary
• Recombinant protein production: bacteria
• Eukaryotic protein production in bacteria
• Problems / solutions
• Site-Directed Mutagenesis – PCR (proof-reading)
• Reporter Genes: examples
• Tags
• Gene Therapy – definitions, different types