Recombinant Growth Factors.

1. Recombinant
Growth Factors
By:
Mohamed Samir El-Asaly
PT, CKTP
Under supervision:
PROF. Dr. Moktar El Zawahri

2. What are growth factors and
what do they do?
 Orderly development requires extensive
coordination and communication between cells.
 Much of this is provided by extracellular proteins
(growth factors) that positively and negatively
regulate proliferation, differentiation,
migration/pathfinding and survival/death.

3. What are growth factors and
what do they do?
 Via transmembrane receptors that transduce growth
factor binding to a cascade of intracellular signaling
events that culminate in both transcription-
independent and transcription-dependent changes in
target cell behavior.
 A number of growth factor superfamilies' have been
recognized along with their specific transmembrane
receptors.

4. What are growth factors and
what do they do?
 Any of a group of proteins that stimulate the growth of
specific tissues. Growth factors play an important role in
promoting cellular differentiation and cell division, and
they occur in a wide range of organisms,
including insects, amphibians, humans, and plants.
 Few of the cellular phenomena that characterize
development are cell autonomous.

5. What are growth factors and
what do they do?
 That is, regulatory molecules that reach cells by an
extracellular route promote and guide virtually every
step of embryogenesis.
 There are on the order of several hundred genes
whose products communicate with cells from the
extracellular space and that influence intracellular
events by binding to specific transmembrane
receptors that in turn transduce such interactions by
activating intracellular signaling pathways.

9. Growth factor families and their
receptors
 There are multiple “superfamilies” of growth factors that contain
multiple subfamilies of proteins, all with related primary sequences.
EXAMPLES OF “CLASSICAL” GROWTH FACTORS
 EGF - EPIDERMAL GROWTH FACTOR
 FGF - FIBROBLAST GROWTH FACTOR
 NGF - NERVE GROWTH FACTOR
 TGFβ - TRANSFORMING GROWTH FACTOR BETA
 INSULIN & IGF’S (INSULIN-LIKE GROWTH FACTORS)
 PDGF- PLATELET DERIVED GROWTH FACTOR

11. Growth factor families and their
receptors
 Such super-families may themselves comprise several
subfamilies, each with multiple sub-members
For instance,
1. The fibroblast growth factor (FGF) superfamily contains at least
22 distinct members.
2. The TGFβ transforming growth factor beta superfamily contains
at least 35 known members that fall into about 10 subfamilies,
one of these subfamilies, the bone morphogenic proteins (BMP’s)
is comprised of at least 15 different gene products.

12. Growth factor families and their
receptors
EXAMPLES OF ADDITIONAL GROWTH FACTOR FAMILIES WITH ROLES IN
DEVELOPMENT
 HEDGEHOG PROTEINS
 WNT’S
 INTERLEUKINS
 SLIT’S
 NETRINS
 EPHRINS
 TUMOR NECROSIS α FAMILY (TNFα’S)

13. Growth factor families and their
receptors
 Each growth factor superfamily has a corresponding family of
related receptors.
 There is high specificity with respect to receptor binding between
super-families.
 But there are cases in which more than one family member binds
to a single receptor and in which a given family member binds to
multiple receptors.
 For instance, there are 4 FGF receptors for the 22 members of
the FGF superfamily.

14. The figure shows binding of the neurotrophin family to their receptors
(designated Trks) and illustrates that a single ligand can bind only a single
receptor, that a given ligand can bind more than one receptor and that one
receptor can bind several different ligands.

15. Growth factor families and their
receptors
Growth factors reach their targets by multiple means
 Long-range dispersion via the circulation (e.g., IGFs)
 “Paracrine” mechanisms of release by local sources (e.g., TGFβ).
 “Autocrine” mechanisms in which a cell responds to growth
factors that it produces itself (e.g., WNTs).
 Direct cell-cell interactions in which the growth factor is itself
presented as a transmembrane protein (e.g., Ephrins).

16. Recombinant human epidermal
growth factors
 Human Epidermal Growth
Factor is a small
polypeptide of molecular
weight 6201 Daltons, with
53 amino acid residues.
 It was discovered by Cohen
(1962)

17. Early work of recombinant EGF
 Several studies prior to 1993 addressed the molecular biology of
recombinant strain construction but EGF yield were low.
 Kim et al. (1992) described low level production of EGF by E.Coli
in continuous culture.
 While Shimizu et al (1991) developed a fed batch procedure for
recombinant EGF production.

18. Early work of recombinant EGF
 In these early studies EGF produced remained inside the microbial cell
 It’s desirable that the Recombinant product can be excreted into the cell
growth medium:
1. Purification would be simpler than for intercellular protein as the
product would not be contaminated with the cytoplasmic components
2. The formation of inclusion bodies would be avoided and possible toxic
effects of EFG peptide product on the host cell would be reduced
3. Excreted protein is stable

19. Early work of recombinant EGF
 In a promising approach, Oka et al (1985) synthetic gene for EGF
was fused with fused signal peptide of E. Coli alkaline phosphate
to direct mature EGF to E. Coli periplasm.
 (1992) Ebisu et al. Bacillus brevis used as the host for
Recombinant EGF. The B. brevis has no external cell wall,
Recombinant EGF may be easily obtained in the culture.
 In this system yield was most impressive (1.1 g/l) but it took (6
days) to reach this level

20. Early work of recombinant EGF
 The long growth time required for EGF production may have an adverse
effect on the activity of the product and would elevate the cost and
prolong the occupancy of the fermentation equipment
 Long fermentation time increase the risk of microbial contamination of
the fermenter which is not acceptable.
 All these deficiencies become even more difficult to tackle in large scale
production

21. Early work of recombinant EGF
 (1993) Promising approach to large scale production of
recombinant EGF.
 Ampicillin resistant E. Coli JM101 strain carrying a Recombinant
EGF encoding plasmid named pWKW2 was produced and
excreted EGF (Wang and Sutherland 1993; Yadward et al. 1994)
with EGF secretion being directed by the E. Coli OmpA signal
sequence.
 The OmpA protein is the major outer membrane protein of E. Coli
and is directed to the outer membrane by a short N terminal
signal sequence, cleaved as protein transverse the inner
membrane of the cell, en route its final destination.

22. EGF gene Cassette
It contains:
• LacUV5 promoter
• Lac operator
• The consensus ribosome binding sequence (RBS) of E. Coli
• OmpA signal sequence
• The Recombinant gene
• Stop Codon
• Stem loop terminator of transcription

23. Plasmid pWKW2
containg EGF gene
 An ampicillin resistance gene
 The ytl2- incR stabilization system
 The EGF encoding gene in the
previous slide is inserted into the
plasmid in which the lacUV5
promoter drives transcription
 The EGF produced in N- terminally
fused to OmapA signal sequence

24. Fermentation
 Fermentation of E.Coli JM101
(pWKW2) with the constant
feeding of medium.
 The working volume was 1 liter,
the inoculum was 10%, the initial
glucose level was 2 g/l, the
fermentation temperature, was
32C and the PH was controlled at
6.8 throughout the fermentation.

25. Scale up of EGF production
 Culture and induction conditions were next optimized with respect
to volumetric production of the EGF
 Fed batch fermentations have been favored for production of
heterologous proteins by recombinant microorganisms.
 Such fermentations afford tight control over environmental
parameter and improve overall product yield compared with
simple batch cultures
 The time course study of a fed batch culture of an induced E. Coli
JM101 starin revealed a gradual increase in the production of EGF
in the first 10 hours post induction to give a yield of 325mg/l.

26. Scale up of EGF production
 The high stability and productivity of this system
facilitated scale up of EGF production and a protocol to
purify EGF from bacterial culture supernatant was
developed by using conventional chromatographic
procedures (Huang et al., 1999) and was shown to be
pure by high pressure liquid chromatography
 The N terminus of the purified hEGF was authentic (
cleavage of the OmpA signal peptide was precise and
degradation from the N terminus was absent or minimal
after excretion of the protein).

27. Synthesis and secretion of human epidermal
growth factor by Escherichia coli
 A synthetic gene for human
epidermal growth factor (hEGF) was
joined to a sequence encoding the
signal peptide of Escherichia coli
alkaline phosphatase.
 This hybrid gene was placed under
the control of the alkaline
phosphatase gene (phoA) promoter
in a recombinant plasmid, which was
used to transfect E. coli.

28. Synthesis and secretion of human epidermal
growth factor by Escherichia coli
 The hybrid protein that was expressed in host cells under
conditions of phosphate limitation was processed accurately
during the secretion process, and mature hEGF was recovered in
the periplasmic fraction.
 On the other hand, no EGF was detected in the periplasmic space
when the synthetic hEGF gene was not accompanied by the phoA
signal sequence.

29. Synthesis and secretion of human epidermal
growth factor by Escherichia coli

30. Synthesis and secretion of human epidermal
growth factor by Escherichia coli

34. Actual and potential uses of
EGF
1. Wound healing.
2. Disturbances in GIT.
3. Targeting of brain and bladder tumors.
4. Minimizing the effects of ureteral obstruction.
5. Assisting the regeneration of nerve tissue.
6. Use in the bioengineering of artificial organs.

35. Actual and potential uses of
EGF
7. Use in repair of eye damage.
8. Assisting the repair of damaged ears.
9. Use in repair of liver injury.
10. Decreasing lung edema.
11. Lowering blood pressure.