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Recombinant growth factors
Growth factors are extracellular proteins that regulate cell proliferation, differentiation, migration, survival and death. They work by binding to transmembrane receptors and activating intracellular signaling pathways. There are several families of growth factors including EGF, FGF, NGF, TGFβ, insulin/IGF and PDGF. Each family has multiple related proteins and receptors. Recombinant human EGF has been produced in E. coli by fusing the EGF gene to a signal sequence to direct secretion, yielding over 300mg/L of purified EGF. Potential uses include wound healing, treating gastrointestinal disturbances and targeting brain/bladder tumors.
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Recombinant growth factors
- 1. Recombinant Growth Factors By: Mohamed SamirEl-Asaly PT, CKTP Under supervision: PROF. Dr. Moktar El Zawahri
- 2. What are growthfactors 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 growthfactors 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 growthfactors 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 growthfactors 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 familiesand 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 familiesand 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 familiesand 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 familiesand 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 showsbinding 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 familiesand 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 growthfactors 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 ofrecombinant 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 ofrecombinant 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 ofrecombinant 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 ofrecombinant 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 ofrecombinant 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 Itcontains: • 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 EGFgene 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 ofE.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 ofEGF 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 ofEGF 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 secretionof 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 secretionof 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 secretionof human epidermal growth factor by Escherichia coli
- 30. Synthesis and secretionof human epidermal growth factor by Escherichia coli
- 34. Actual and potentialuses 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 potentialuses 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.