An efficient system for high‐level expression and easy purification of authentic recombinant proteins

Catanzariti, Ann‐Maree; Soboleva, Tatiana; Jans, David A.; Board, Philip G.; Baker, Rohan T.

doi:10.1110/ps.04618904

Cited by 276 publications

(295 citation statements)

References 27 publications

(33 reference statements)

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“…The AvrL567-A, AvrL567-C, and AvrL567-D proteins were expressed in E. coli strain BL21(DE3) as ubiquitin fusion proteins containing an Nterminal hexahistidine tag by using the vector pHUE (34). Cultures were grown at 37°C to a OD 600 of Ϸ0.8-1.0 in LB broth and induced with 1 mM isopropyl thio-␤-D-galactoside for 18-21 h at 15°C.…”

Section: Methodsmentioning

confidence: 99%

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

Dodds

Lawrence

Catanzariti

et al. 2006

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

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694

520

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Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R-Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R-Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R-Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant-pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.avirulence protein ͉ resistance protein P lants resist disease through a variety of preformed and induced barriers to infection. Among these is a genetically determined pathogen recognition system controlled by host resistance genes (R genes). In this gene-for-gene resistance system, plant R genes confer resistance to pathogen strains carrying corresponding avirulence (Avr) genes (so-called because their presence prevents growth on resistant plants). The recognition event involving the products of the R and Avr genes triggers host defense responses, including a localized host cell death or hypersensitive response (HR) that limits the spread of the pathogen from the infection site. Most known plant resistance proteins (R proteins) contain nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains, with the latter implicated in pathogen recognition (1). In contrast, Avr proteins are diverse, and many have pathogenicity effector functions that play important roles in enhancing infection (2). The antagonistic relationship between R and Avr genes results in coevolutionary conflict as selection favors evolution of resistance in plants and virulence in their pathogens.The Arabidopsis RPM1, RPS2, and RPS5 NBS-LRR-type R proteins confer r...

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Section: Methodsmentioning

confidence: 99%

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

Dodds

Lawrence

Catanzariti

et al. 2006

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

Self Cite

694

520

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“…The pHUE-EAS plasmid (15,48) was used as a template to generate the required EAS mutants via either standard or two-step overlap extension PCR. Synthetic genes encoding NC2 and the chimera were purchased from Genscript and subcloned into the pHUE vector (15).…”

Section: Methodsmentioning

confidence: 99%

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Macindoe

Kwan

Ren

et al. 2012

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

120

111

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The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-β rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.A myloid fibrils were first identified in association with human diseases, but recent discoveries show that the amyloid ultrastructure also contributes to important functions in normal biology (1, 2). In bacteria, fungi, insects, fish, and mammals, amyloid structures perform a wide variety of roles (3). Functional amyloids in the form of fibrillar rodlets composed of class I hydrophobin proteins are found in filamentous fungi. These hydrophobins are small proteins that are secreted as monomers and self-assemble into rodlets that pack to form amphipathic monolayers at hydrophilic: hydrophobic boundaries, such as the surface of the growth medium (4). These proteins are extremely surface active and lower the surface tension of the aqueous growth medium, allowing hyphae to break through the surface and to produce aerial structures (5, 6). Many of these aerial structures subsequently become coated with amyloid rodlets, creating a hydrophobic layer that serves multiple purposes, including conferring water resistance to spores for easier dispersal in air (7), preventing wetting or collapse of gas transfer channels (8), enhancing adherence to waxy surfaces such as leaves during infection of rice plants by Magnaporthe grisea (9), and mediating evasion of the immune system as is observed in Aspergillus fumigatus infections (10).Hydrophobins are characterized by the presence of eight cysteine residues that form four disulphide bonds, but the hydrophobin family can be further divided into two classes based on the spacing of the conserved cysteine residues and the nature of the amphipathic monolayers that they form (11). Class I, but not class II, hydrophobins form amyloid-like rodlets that are extremely robust and require treatment with strong acid to induce depolymerization. The amphipathic monolayers formed by class II hydrophobins are not fibrillar and can be dissociated by treatment with detergent and alcohol solutions. The soluble, monomeric forms of hydrophobins share a unique β-barrel topology, and all have a relatively large exposed hydrophobic area on the protein monomer surface (4). The diversity in sequence and chain length ...

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“…Proteins were produced as His 6 -ubiquitin fusion using the Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by genesdev.cshlp.org Downloaded from pHUE vector system as described previously (Catanzariti et al 2004;Baker et al 2005).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

et al. 2014

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The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed b propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

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An efficient system for high‐level expression and easy purification of authentic recombinant proteins

Cited by 276 publications

References 27 publications

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

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