1.
Extraction from natural system
Required large quantities
80 μg of, the pure and biologically-active human hormone
secretin, would require 3000 kg of bovine intestine.
Difficult to meet the demand
The quantity of purified insulin obtained from one pig
satisfies the requirements of one diabetic person for three day
Contamination

2.
Commercial production of gene products requires
very high expression levels
Factors that affect gene expression can be manipulated to
increase the amount of protein production
 Move gene to specialized expression vectors and hosts
 Location of gene (vector or host's chromosome)
 Gene dosage
 Transcription
• Promoter and terminator sequences
• Regulatory genes and sequences

3.
 Translation
• Ribosome binding site (Shine-Delgarno sequence)
• Codon optimization to match host's codon bias
 Final location of gene product
• Cytoplasmic or extracellular (secreted out of cell)
 Protein stability
• Degradation by host proteases (fusion protein)
• Formation of insoluble aggregates

4.
Transcription
Promoters and enhancers are two major regulatory regions
that controlled the transcription.
Promoter contains specific DNA sequences that act as
‘molecular switches’ to turn on transcription, in the upstream
direction of the genes.
Eukaryotic genes have enhancers in addition to promoters.
Enhancers enhances transcription and are functional in any
orientation.

5.
Types of promoter to regulate gene expression
Constitutive promoters
Direct expression in virtually all tissues and are largely, if not
entirely, independent of environmental factors.
Tissue-specific promoters
Direct expression of a gene in specific tissue or at certain
stages of development.
Chemical-inducible promoters
Their performance can be controlled artificially
Synthetic promoters
Made of the primary elements of a promoter region

6.
The most commonly used promoters in E. coli

7.
Translation
The ribosome binding site (RBS)
• Vary between E. coli strains
• A consensus sequence (Shine-Delgarno sequence) is
centered ~ 10 nucleotides upstream from the start codon.
• Most sites contain the minimal sequence ***AGGA****
• Base pairing between a mRNA’s Shine Delgarno sequence
and the ribosome to select the proper initiation codon
• If a eukaryotic gene to be expressed in E.coli, is cloned
starting from the its first codon, a Shine-Delgarno sequence
will have to be inserted upstream for the translation to occur

8.
Codon Optimization

9.
Final Yield of Protein
Expression of protein in secretion vector
Proteins in periplasm easier to purify
Protein often more stable in periplasm
Secretion to periplasm requires signal sequence

10.
Protein stability: Fusion Proteins
Recover from
DNA culture medium
and purify
peptide
protein of interest
mRNA Maltose-binding protein

11.
Fusion Proteins

12.
Poor expression ?
Presence of introns in eukaryotes : has been solved by using cDNA
prepared from mRNA
Degradation of foreign proteins: prevented by using protease
deficient strain or by fusion protein technique.
Incorrect folding:
leading to insoluble protein aggregates (inclusion bodies).
Fusing to E. coli protein thioredoxin can increase solubility up to 40%
of total cellular protein

13.
Insoluble proteins are not a curse
The blessings of inclusion bodies
• Easy to segregate proteins in inclusion bodies by centrifugation.
• Inclusion bodies protect protein from proteases.
• Toxic proteins won’t kill the host cells before enough is
expressed to be useful.
On the other hand
• Protein must be dissolved out of the inclusion bodies to be
purified.
• Inclusion body proteins are usually not folded completely;
finishing the job can be a challenge.
• Refolding is seldom complete and the portion of the protein
which is active may be very low at best.

14.
Problems with E. coli
Endotoxins contamination
Not able to do post-translational modifications like
Glycosylation
Required for,
Fatty acid acylation Proper folding of the
secondary, tertiary and
Phosphorylation and
quaternary structures of
Disulfide-bond formation interested proteins
These modifications can affect the
bioactivity, function, structure, solubility, stability, half-life,
protease resistance, and compartmentalization of the
functional proteins.