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Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in
J Hoppe1, H A Weich, W Eichner
1Department of Cytogenetics, GBF--Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, FRG.
Abstract:
Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.
Insights
Researchers produced biologically active recombinant platelet-derived growth factor BB (rPDGF-BB) in E. coli. This recombinant growth factor stimulated fibroblast proliferation, demonstrating its mitogenic potential for research applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Platelet-derived growth factor (PDGF) is a mitogen composed of A and B chains.
- PDGF-B shares homology with the simian sarcoma virus transforming protein (p28v-sis).
Purpose of the Study:
- To express and characterize a PDGF-BB-like homodimer in Escherichia coli.
- To assess the mitogenic activity of the recombinant PDGF-BB.
Main Methods:
- Engineered E. coli to express a cro-beta-gal-PDGF-B fusion protein.
- Purified monomeric PDGF-B fragment using CNBr cleavage, S-sulfonation, and chromatography.
- Dimerized the monomeric fragment to produce biologically active rPDGF-BB.
Main Results:
- Obtained approximately 0.7 mg of rPDGF-BB per liter of E. coli culture.
- rPDGF-BB demonstrated significant mitogenic activity, stimulating [3H]thymidine incorporation in AKR2B fibroblasts at ~1 ng/mL.
Conclusions:
- Successfully produced biologically active recombinant PDGF-BB in E. coli.
- The recombinant PDGF-BB exhibits potent mitogenic effects comparable to native PDGF.