Separation of truncated basic fibroblast growth factor from the full-length protein by hydrophobic interaction chromatography

Sauer, Dominik Georg; Mosor, Magdalena; Jungbauer, Alois; Dürauer, Astrid

doi:10.1016/j.seppur.2020.117564

Cited by 7 publications

(3 citation statements)

References 28 publications

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“…In addition, for PRbFGF, a smaller truncated bFGF band was seen being generated in the CBB staining of the crude extracts as incubation time progressed (Figure 4B, lower panel and Supplementary Figure S2). A similar formation of this truncated form was reported for purified bFGF (Sauer et al 2021).…”

Section: Stability Analysis Of Prbfgf and Bfgf In Crude Extractssupporting

confidence: 84%

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in Nicotiana benthamiana leaves

Macauyag

Kajiura

Ohashi

et al. 2022

Plant Biotechnology

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The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium-mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 µg g −1 FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli-produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts.

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Section: Stability Analysis Of Prbfgf and Bfgf In Crude Extractssupporting

confidence: 84%

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in Nicotiana benthamiana leaves

Macauyag

Kajiura

Ohashi

et al. 2022

Plant Biotechnology

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show abstract

“…To ensure the absence of host cell-derived proteins that could otherwise lead to erroneous results in antibody tests, additional unit operations are usually performed after capture, such as ion exchange or hydrophobic interaction chromatography. HIC is a potent purification method to separate highly similar proteins, such as protein variants or fragments [ 31 , 32 ]. This purification step also aids in removal of host cell proteins still present after IMAC [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection

Vos

Aguilar

Köppl

et al. 2021

Talanta

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The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency for dimerization and interaction with nucleic acids. For the first time, high titers of NP were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system . Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of downstream processing (DSP) steps. An analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77% after cell lysis. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The optimized HIC-MALS method enables monitoring of the product purity, and simultaneously access its molecular mass, providing orthogonal information complementary to established SEC-MALS methods. Enhanced resolving power can be achieved over SEC, attributed to the extended variables to tune selectivity in HIC mode.

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“…To ensure the absence of host cell-derived proteins that could otherwise lead to erroneous results in antibody tests, additional unit operations are usually performed after capture, such as ion exchange or hydrophobic interaction chromatography. HIC is a potent purification method to separate highly similar proteins, such as protein variants or fragments 30,31 . This purification step also aids in removal of host cell proteins still present after IMAC 32 .…”

Section: Introductionmentioning

confidence: 99%

Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

Vos

Aguilar²,

Köppl³

et al. 2021

Preprint

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The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. <a>This protein has a strong tendency to dimerization and interaction with nucleic acids. A native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) method was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. High titers of the nucleocapsid protein were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of chromatographic steps</a>. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77%. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The HIC-MALS method enables to monitor the purity of the product and simultaneously access its molecular mass.

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Separation of truncated basic fibroblast growth factor from the full-length protein by hydrophobic interaction chromatography

Cited by 7 publications

References 28 publications

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in <i>Nicotiana benthamiana</i> leaves

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in <i>Nicotiana benthamiana</i> leaves

Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection

Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

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