Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography

Müller, Carsten; Rinas, Ursula

doi:10.1016/s0021-9673(99)00660-3

Cited by 40 publications

(30 citation statements)

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“…Production of PDGF was carried out in a high cell density cultivation procedure that has been described previously (24). Purification of unfolded and reduced PDGF monomers from solubilized PDGF containing inclusion bodies was done by size exclusion chromatography (SEC) under denaturing conditions (14).…”

Section: Production and Purification Of Pdgf Isoforms-mentioning

confidence: 99%

“…Kinetic Analysis of PDGF Dimerization-Purified, unfolded, and reduced PDGF monomers (either pure A-, or B-chains, or an equimolar mixture of the two chains) were subjected to SEC under conditions that allow refolding and reshuffling of disulfide bridges as described previously (14). Standard renaturation conditions by SEC were: 1 mol l…”

Section: Production and Purification Of Pdgf Isoforms-mentioning

confidence: 99%

“…However, the formation of the cystine knot appears to be indispensable for the biological activity of PDGF (21), while the intermolecular disulfide bridges have a stabilizing but non-essential effect on the biological activity (21,22). PDGF is very prone to aggregation when renaturation is initiated by diluting unfolded and reduced monomers into a buffer, which allows refolding and disulfide bond reshuffling (14). Once folded, however, PDGF is a very stable protein withstanding temperatures of up to 100°C (23).…”

mentioning

confidence: 99%

“…Previously, we have presented a renaturation method based on the utilization of size exclusion chromatography, which circumvents aggregation during refolding and allows renaturation of PDGF at high protein concentrations (14). In this study, we present a kinetic analysis of the formation of disulfide-bonded dimers and propose a model for the folding pathway of the different isoforms of PDGF.…”

mentioning

confidence: 99%

“…Under these conditions, the eluted PDGF monomers were able to dimerize in the eluate fraction to yield the dimeric, disulfide-bonded, and biologically active growth factor (14). Aggregation of PDGF during the renaturation procedure was not observed unless otherwise indicated.…”

mentioning

confidence: 99%

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Kinetics Control Preferential Heterodimer Formation of Platelet-derived Growth Factor from Unfolded A- and B-chains

Müller¹,

Richter²,

Rinas³

2003

Journal of Biological Chemistry

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The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in woundhealing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A-or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfidebonded heterodimers from an equimolar mixture of unfolded A-and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.

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Section: Production and Purification Of Pdgf Isoforms-mentioning

confidence: 99%

Section: Production and Purification Of Pdgf Isoforms-mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetics Control Preferential Heterodimer Formation of Platelet-derived Growth Factor from Unfolded A- and B-chains

Müller¹,

Richter²,

Rinas³

2003

Journal of Biological Chemistry

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Refolding of Inclusion Body Proteins from E. coli

Su¹,

Lu²,

Liu³

2011

Methods of Biochemical Analysis

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Optimized procedure for renaturation of recombinant human bone morphogenetic protein‐2 at high protein concentration

Vallejo

Rinas

2004

Biotech & Bioengineering

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The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.

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Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography

Cited by 40 publications

References 28 publications

Kinetics Control Preferential Heterodimer Formation of Platelet-derived Growth Factor from Unfolded A- and B-chains

Kinetics Control Preferential Heterodimer Formation of Platelet-derived Growth Factor from Unfolded A- and B-chains

Refolding of Inclusion Body Proteins from E. coli

Optimized procedure for renaturation of recombinant human bone morphogenetic protein‐2 at high protein concentration

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