Characterization of a dockerin‐based affinity tag: application for purification of a broad variety of target proteins

Demishtein, Alik; Karpol, Alon; Barak, Yoav; Lamed, Raphael; Bayer, Edward A.

doi:10.1002/jmr.1029

Cited by 23 publications

(17 citation statements)

References 40 publications

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“…S1A. The construct is denoted xylanase-dockerin, and a version without the T129C mutation had been produced and characterized in previous works by our group (4,6). Protein amino acid sequences are provided in Dataset S1.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule dissection of the high-affinity cohesin–dockerin complex

Ståhl

Nash

Fried³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for selfassembly of the cellulosomal components into the multienzyme complex. The observed cohesin-dockerin rupture forces (>120 pN) are among the highest reported for a receptor-ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop-helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin's critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein-protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines. molecular recognition | protein unfolding

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Section: Resultsmentioning

confidence: 99%

Single-molecule dissection of the high-affinity cohesin–dockerin complex

Ståhl

Nash

Fried³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

114

128

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“…Phosphoric acid-swollen cellulose (PASC) was prepared according to Lamed et al [9]. GFP-CBM3a was prepared according to Demishtein et al [10].…”

Section: Methodsmentioning

confidence: 99%

Glycoside hydrolases as components of putative carbohydrate biosensor proteins in Clostridium thermocellum

Bahari¹,

Gilad²,

Borovok³

et al. 2010

J Ind Microbiol Biotechnol

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The composition of the cellulase system in the cellulosome-producing bacterium, Clostridium thermocellum, has been reported to change in response to growth on different carbon sources. Recently, an extensive carbohydrate-sensing mechanism, purported to regulate the activation of genes coding for polysaccharide-degrading enzymes, was suggested. In this system, CBM modules, comprising extracellular components of RsgI-like anti-σ factors, were proposed to function as carbohydrate sensors, through which a set of cellulose utilization genes are activated by the associated σ(I)-like factors. An extracellular module of one of these RsgI-like proteins (Cthe_2119) was annotated as a family 10 glycoside hydrolase, RsgI6-GH10, and a second putative anti-σ factor (Cthe_1471), related in sequence to Rsi24, was found to contain a module that resembles a family 5 glycoside hydrolase (termed herein Rsi24C-GH5). The present study examines the relevance of these two glycoside hydrolases as sensors in this signal-transmission system. The RsgI6-GH10 was found to bind xylan matrices but exhibited low enzymatic activity on this substrate. In addition, this glycoside hydrolase module was shown to interact with crystalline cellulose although no hydrolytic activity was detected on cellulosic substrates. Bioinformatic analysis of the Rsi24C-GH5 showed a glutamate-to-glutamine substitution that would presumably preclude catalytic activity. Indeed, the recombinant module was shown to bind to cellulose, but showed no hydrolytic activity. These observations suggest that these two glycoside hydrolases underwent an evolutionary adaptation to function as polysaccharide binding agents rather than enzymatic components and thus serve in the capacity of extracellular carbohydrate sensors.

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“…thermocellum , may be the result of one or more effects. First, while the cohesin dockerin interaction is quite robust, it is possible that when produced by two different strains the assembly of mature cellulosomes occurs less efficiently than if the components are being secreted simultaneously from the same cell [39,40]. Second, the assembled cellulosomes are incapable of adhering to the surface of the more metabolically active T .…”

Section: Discussionmentioning

confidence: 99%

Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

Currie

Herring

Guss

et al. 2013

Biotechnol Biofuels

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BackgroundCellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes.ResultsWe developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome.ConclusionThis study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.

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Characterization of a dockerin‐based affinity tag: application for purification of a broad variety of target proteins

Cited by 23 publications

References 40 publications

Single-molecule dissection of the high-affinity cohesin–dockerin complex

Single-molecule dissection of the high-affinity cohesin–dockerin complex

Glycoside hydrolases as components of putative carbohydrate biosensor proteins in Clostridium thermocellum

Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

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