Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Lahtinen, Tomi; Linder, Markus B.; Nakari-Setälä, Tiina; Oker‐Blom, Christian

doi:10.1016/j.pep.2007.12.014

Cited by 34 publications

(35 citation statements)

References 28 publications

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“…More recently, the utility of this ATPS technology was demonstrated for recovering hydrophobin fusions from insect cell extracts (Lahtinen et al, 2008). To our knowledge, fungal hydrophobins have not been previously expressed in plants, so we evaluated the effectiveness of HFBI as a fusion partner for one-step ATPS purification in plants.…”

Section: Discussionmentioning

confidence: 99%

“…Acidic sodium acetate buffers (pH 5.0-5.5) have been used successfully with ATPSs Lahtinen et al, 2008), but these buffers offered a low recovery when attempting to extract GFP-HFBI from the N. benthamiana leaves (data not shown). This result can likely be explained by the precipitation of many plant proteins at a pH below 6 (Joensuu et al, 2009) and by the instability of GFP at an acidic pH (Patterson et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…Although hydrophobins have been used as purification tags in ATPS Lahtinen et al, 2008), they have not been previously used to express or purify plant-made proteins. Therefore, we optimized the ATPS separation of GFP and GFP-HFBI leaf extracts using three different concentrations (2%, 4%, and 8% [w/v]) of the surfactant Agrimul NRE 1205.…”

Section: Purification Of Gfp-hfbi By An Atpsmentioning

confidence: 99%

“…The ATPS concentrates the hydrophobin fusions inside micellar structures and partitions them toward the surfactant phase (Lahtinen et al, 2008). ATPSs offer several benefits, since they are simple, rapid, and inexpensive while providing volume reduction, high capacity, and fast separations (Persson et al, 1999).…”

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Hydrophobin Fusions for High-Level Transient Protein Expression and Purification inNicotiana benthamiana

et al. 2009

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Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL 21 . HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Purification Of Gfp-hfbi By An Atpsmentioning

confidence: 99%

mentioning

confidence: 99%

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Hydrophobin Fusions for High-Level Transient Protein Expression and Purification inNicotiana benthamiana

et al. 2009

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“…Hydrophobins can solubilize and reverse the wettability of nanomaterials such as carbon nanotubes (19 -21), be used to functionalize surfaces and be applied for the two-phase separation of proteins (22,23). Class I hydrophobin monolayers also have been shown to be immunologically inert (24), and together with the ability of hydrophobins to form suspensions with water-insoluble moieties in biological solutions, this has prompted interest in the use of hydrophobins as drug-delivery agents (25,26).…”

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Recruitment of Class I Hydrophobins to the Air:Water Interface Initiates a Multi-step Process of Functional Amyloid Formation

Morris

Ren

Macindoe

et al. 2011

Journal of Biological Chemistry

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Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.Class I hydrophobins are a family of small amphipathic proteins that are produced by filamentous fungi in a monomeric form but are able to self-assemble into amphipathic monolayers composed of amyloid-like structures known as rodlets (1-3). The polymerization of the hydrophobins occurs on contact with a hydrophobic:hydrophilic interface, such as an air:water boundary or when hydrophobins are secreted from the spores and come into contact with the air. Members of the hydrophobin family are characterized by the presence of four disulfide bonds. The amphipathic nature of hydrophobins drives them to the surface of solutions, where they reduce the surface tension (1).Some of the functional roles of the class I hydrophobins include acting as a surfactant at the air:water boundary to reduce the surface tension, which is a barrier to aerial growth of hyphae, and also to form a robust protein coat on spores. This coating provides a hydrophobic external surface that resists wetting and thus facilitates spore dispersal in air (4 -6). The class I hydrophobin rodlets share many of the structural characteristics of amyloid fibrils; formation of the insoluble, fibrillar rodlets is accompanied by conformational change to an ordered cross-␤-secondary structure form and the polymerized rodlets, but not the monomeric form of the protein, bind to the dye thioflavin T (ThT) 4 (2, 7, 8). However, in contrast to other amyloid fibrils, which can often be solubilized by treatment with denaturants such as guanidine hydrochloride or solvents such as dimethyl sulfoxide (9), treatment with acids such as formic and TFA has been reported to be the only method capable of depolymerizing hydrophobin rodlets and regenerating the monomeric form of the hydrophobin proteins in solution (10,11). This is similar to the curli and tafi fibrils produced by bacteria, which have been shown to be functional amyloid and which also req...

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Rhizosecretion improves the production of Cyanovirin‐N in Nicotiana tabacum through simplified downstream processing

Madeira

Szeto

K‐C.

et al. 2016

Biotechnology Journal

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Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N.

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Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Cited by 34 publications

References 28 publications

Hydrophobin Fusions for High-Level Transient Protein Expression and Purification inNicotiana benthamiana

Hydrophobin Fusions for High-Level Transient Protein Expression and Purification inNicotiana benthamiana

Recruitment of Class I Hydrophobins to the Air:Water Interface Initiates a Multi-step Process of Functional Amyloid Formation

Rhizosecretion improves the production of Cyanovirin‐N in Nicotiana tabacum through simplified downstream processing

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