Intracellular protein production in Trichoderma reesei (Hypocrea jecorina) with hydrophobin fusion technology

Mustalahti, Eero; Saloheimo, Markku; Joensuu, Jussi

doi:10.1016/j.nbt.2011.09.006

Cited by 22 publications

(16 citation statements)

References 38 publications

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“…Hydrophobins are small surface proteins (7-12 KDa) enrolled in numerous functions on filamentous fungi life cycle. The unique properties of these small proteins are related to their amphiphilic nature and selfassembly capacity (Mustalahti et al, 2013). There are two classes (I and II) of hydrophobins differing on the solubility of the formed aggregates and the amino acid composition.…”

Section: Hydrophobinmentioning

confidence: 99%

“…In terms of aggregates solubility, class I forms more stable aggregates than class II and therefore, in the first case, the aggregates are only able to dissociate under the presence of strong conditions (e.g. formic acid and trifluoroacetic acid) whilst the aggregates formed by class II hydrophobins are much easier to dissolve with aqueous diluted organic solvents (Linder et al, 2005;Mustalahti et al, 2013).…”

Section: Hydrophobinmentioning

confidence: 99%

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Challenges and opportunities in the purification of recombinant tagged proteins

Pina

Lowe

Roque

2014

Biotechnology Advances

121

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The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development.

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Section: Hydrophobinmentioning

confidence: 99%

Section: Hydrophobinmentioning

confidence: 99%

Challenges and opportunities in the purification of recombinant tagged proteins

Pina

Lowe

Roque

2014

Biotechnology Advances

121

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“…Carrier fusions of this type have been proposed to have a diversely positive effect on production levels by stabilising mRNA, improving intracellular trafficking in the secretory pathway, and proximity shielding against proteolysis [9]. An alternative strategy can be to direct intracellular accumulation of a target protein, previously highlighted by the hydrophobin fusion strategy leading to protein body (PB) formation [10,11]. Another self-assembling protein that can promote intracellular accumulation and PB formation is derived from the maize gamma zein storage protein [12], ZERA®.…”

Section: Introductionmentioning

confidence: 99%

Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

et al. 2014

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BackgroundTrichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling.ResultsConstructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a method was developed for gravity-based density separation and protein body solubilisation. The secreted protein had similar specific activity to that of the commercially available mammalian form. The intracellular version had 5-10-fold lower activity due to the enzymes incompatibility with alkaline pH. The secretion strain achieved 10% lower total biomass than either the parental or the intracellular strain. The patterns of gene induction for intracellular and parental strains were similar, whereas the secretion strain had a broader spectrum of gene expression level changes. Identification of the genes involved indicated strong secretion stress in the secretion strain and to a lesser extent also in intracellular production. Genes involved in the unfolded protein response (UPR) and ER-associated degradation were induced by GLA production, including; hac1, pdi1, prp1, cnx1, der1, and bap31.ConclusionsActive human α-galactosidase could most effectively be produced intracellularly in Trichoderma reesei at >0.5 g/l by avoidance of the extracellular environment, although purification was challenging due to specific activity losses. Strain analysis revealed that in addition to the issues with secreted proteases, the processes of secretion stress including UPR and ER degradation remain as bottlenecks for heterologous protein production. Genetic engineering to eliminate these bottlenecks is the logical path towards establishing a strain capable of producing sensitive heterologous proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0091-y) contains supplementary material, which is available to authorized users.

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“…Another case clearly showed that targeted insertion of a 652 glucoamylase gene from Hormoconis resinae into the cbh1 locus yielded 653 3-fold higher production than the same gene randomly inserted in trip-654 licate in the genome (Joutsjoki, 1994). This work was complicated by 655 the use of gDNA for the targeted insertion and cDNA for the random in-656 sertion; however, both DNA forms gave folded and active protein, 657 indicating that intron processing was carried out correctly for the heter- or mus53 (Guangtao et al, 2009;Mustalahti et al, 2013;Schuster 670 et al, 2012a;Steiger et al, 2011).…”

mentioning

confidence: 99%

Heterologous protein expression in Hypocrea jecorina: A historical perspective and new developments

Singh

Taylor

Wall

et al. 2015

Biotechnology Advances

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Hypocrea jecorina, the sexual teleomorph of Trichoderma reesei, has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or improved variants of native enzymes. Expression of heterologous proteins in H. jecorina was once considered difficult when the target was an improved variant of a native cellulase. Developments over the past nearly 30 years have produced strains, vectors, and selection mechanisms that have continued to simplify and streamline heterologous protein expression in this fungus. More recent developments in fungal molecular biology have pointed the way toward a fundamental transformation in the ease and efficiency of heterologous protein expression in this important industrial host. Here, 1) we provide a historical perspective on advances in H. jecorina molecular biology, 2) outline host strain engineering, transformation, selection, and expression strategies, 3) detail potential pitfalls when working with this organism, and 4) provide consolidated examples of successful cellulase expression outcomes from our laboratory.

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Intracellular protein production in Trichoderma reesei (Hypocrea jecorina) with hydrophobin fusion technology

Cited by 22 publications

References 38 publications

Challenges and opportunities in the purification of recombinant tagged proteins

Challenges and opportunities in the purification of recombinant tagged proteins

Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

Heterologous protein expression in Hypocrea jecorina: A historical perspective and new developments

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