Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

Gospodarek, Adrian; Sun, Wenjun; O’Connell, John P.; Fernandez, Erik J.

doi:10.1016/j.chroma.2014.10.080

Cited by 6 publications

(3 citation statements)

References 50 publications

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“…It is well accepted that protein adsorption and desorption may lead to conformational changes and thus alter protein bioactivity. , The prerequisite for the utilization of any delivery system is the release of BMP-2 maintaining high bioactivity. Thus, the biological activity of BMP-2 released from SCH-D and SCH-L scaffolds was first determined by alkaline phosphatase (ALP) staining and matrix mineralization deposition.…”

Section: Resultsmentioning

confidence: 99%

Mesoporous Hydroxyapatite Nanoparticles Mediate the Release and Bioactivity of BMP-2 for Enhanced Bone Regeneration

Qiu

Guo

et al. 2020

ACS Biomater. Sci. Eng.

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Efficient delivery of bone morphogenetic protein-2 (BMP-2) with desirable bioactivity is still a great challenge in the field of bone regeneration. In this study, a silk fibroin/chitosan scaffold incorporated with BMP-2-loaded mesoporous hydroxyapatite nanoparticles (mHANPs) was prepared (SCH-L). BMP-2 was preloaded onto mHANPs with a high surface area before mixing with a silk fibroin/chitosan composite. Bare (without BMP-2) silk fibroin/chitosan/mHANP (SCH) scaffolds and SCH scaffolds with directly absorbed BMP-2 (SCH-D) were investigated in parallel for comparison. In vitro release kinetics indicated that BMP-2 released from the SCH-L scaffold showed a significantly lower initial burst release, followed by a more sustained release over time than the SCH-D scaffold. In vitro cell viability, osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and the in vivo osteogenic effect of scaffolds in a rat calvarial defect were evaluated. The results showed that compared with bare SCH and SCH-D scaffolds, the SCH-L scaffold significantly promoted the osteogenic differentiation of BMSCs in vitro and induced more pronounced bone formation in vivo. Further studies demonstrated that the mHANP-mediated satisfactory conformational change and sustained release benefited the protection of the released BMP-2 bioactivity, as confirmed by alkaline phosphatase (ALP) activity and a mineralization deposition assay. More importantly, the interaction of BMP-2/mHANPs enhanced the binding ability of BMP-2 to cellular receptors, thereby maintaining its biological activity in osteogenic differentiation and osteoinductivity well, which contributed to the markedly promoted in vitro and in vivo osteogenic efficacy of the SCH-L scaffold. Taken together, these results provide strong evidence that mHANPs represent an attractive carrier for binding BMP-2 to scaffolds. The SCH-L scaffold shows promising potential for bone tissue regeneration applications.

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Section: Resultsmentioning

confidence: 99%

Mesoporous Hydroxyapatite Nanoparticles Mediate the Release and Bioactivity of BMP-2 for Enhanced Bone Regeneration

Qiu

Guo

et al. 2020

ACS Biomater. Sci. Eng.

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“…Whilst the concepts related to the effects of different salt types and concentrations on the retention of proteins in hydrophobic interaction chromatography (HIC) have been elaborated over the past decade, [1][2][3] there are still gaps of knowledge concerning the correlation between the chromatographic behaviour of proteins and their conformational structures immediately following elution. Small Angle X-ray Scattering (SAXS) is one of the most powerful techniques to determine protein size and shape in solution and in the solid state.…”

Section: Introductionmentioning

confidence: 99%

On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column

Kulsing

Komaromy

Boysen

et al. 2016

Analyst

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This study documents the use of an integrated approach, involving on-line hydrophobic interaction chromatography interfaced with Small Angle X-ray Scattering (HIC-SAXS) measurements, to monitor the conformational status of proteins immediately upon elution from a chromatographic column operated at different temperatures. Moreover, this approach provides an additional avenue to interrogate the changes in protein shape that may occur across the eluted chromatographic peak. To this end, radii of gyration were extrapolated from the Guinier approximation with the HIC-SAXS data, whilst pair distribution functions and bead model simulations were generated by using the indirect transform program GNOM and ab initio reconstruction with GASBOR to provide further insight into protein conformational changes that occur during hydrophobic interaction chromatography.

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“…HPHIC plays important roles in protein refolding with purification . For example, when denatured protein is loaded onto the HPHIC column using 8.0 M urea in a specific linear gradient elution mode, stretch polypeptide chains are first adsorbed onto the HPHIC stationary phase at high salt concentrations with removal of the denaturing agent and then desorbed at low salt concentrations . During the adsorption and desorption processes, denatured proteins can be folded into a natural state or other folding intermediates , depending on the match between the hydrophobic region of the polypeptide chain and the hydrophobic groups in the HPHIC stationary phase and also on the structure of the ligands .…”

Section: Introductionmentioning

confidence: 99%

Preparation of a silica‐based high‐performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation

Yang

Liu

et al. 2016

J of Separation Science

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In this work, based on the structural characteristics of bio-membrane molecules, a novel type of high-performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one-step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.

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Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

Cited by 6 publications

References 50 publications

Mesoporous Hydroxyapatite Nanoparticles Mediate the Release and Bioactivity of BMP-2 for Enhanced Bone Regeneration

Mesoporous Hydroxyapatite Nanoparticles Mediate the Release and Bioactivity of BMP-2 for Enhanced Bone Regeneration

On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column

Preparation of a silica‐based high‐performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation

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