Cloning, expression, and deletion analysis of large nanH of Clostridium perfringens ATCC 10543

Sheu, Sheh Yi; Tseng, Huen Juin; Huang, Shu Ping; Chien, Chin Hsiang

doi:10.1016/s0141-0229(02)00177-1

Cited by 5 publications

(5 citation statements)

References 19 publications

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“…Previous studies had subcloned residues 216±694 and found that this domain exhibited a similar speci®c activity to the full-length protein (Sheu et al, 2002). From its sequence similarity to the leech sialidase, the positions of the conserved RIP and Asp-box motifs and the conservation of other conserved active-site residues (Taylor, 1996), we can assume that all of the catalytic activity resides in the 243± 694 domain.…”

Section: Resultsmentioning

confidence: 84%

“…This revealed a 50.3 kDa protein with an N-terminus beginning VEGA, corresponding to residues 243±694. The fulllength protein was prone to degradation (Sheu et al, 2002), which we were unable to prevent and which led to problems in reproducibility of crystals. We therefore decided to subclone the catalytic domain.…”

Section: Expression Purification and Crystallization Of Nanimentioning

confidence: 96%

“…Full-length nanI from C. perfringens ATCC 10543 with a six-histidine tag at the C-terminus was expressed and puri®ed as described previously (Sheu et al, 2002). Crystals were grown at 293 K under oil using a 96-well microbatch system and a Douglas Instruments robot with 2 ml protein solution and 2 ml precipitant solution.…”

Section: Expression Purification and Crystallization Of Nanimentioning

confidence: 99%

“…The fragment of the gene encoding the C-terminal catalytic domain (amino acids 243±694) was ampli®ed by PCR from vector DNA encoding the full-length nanI sialidase gene (Sheu et al, 2002). Primers were designed to create an NcoI site incorporating the initition codon at the 5 H site and a PstI site following the termination codon.…”

Section: Subcloning Expression and Purificationmentioning

confidence: 99%

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Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, fromClostridium perfringens

Newstead

Chien²,

Taylor

et al. 2004

Acta Crystallogr D Biol Cryst

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Sialidases catalyse the removal of terminal sialic acids from a range of glycoproteins, glycolipids and oligosaccharides. They have been found in bacteria, viruses and parasites, where they play important roles in pathogenesis and/or microbial nutrition, and in mammalian cells, where they modulate cell-surface glycosylation associated with a range of cellular activities. Clostridium perfringens, a causative agent of gas gangrene and peritonitis in humans, possesses three sialidases: nanH, nanI and nanJ, with molecular weights of 42, 77 and 129 kDa, respectively. The two larger enzymes are secreted by the bacterium and are involved in the pathogenesis and nutrition of Clostridium. As part of a study to examine the structures of all three enzymes, crystallization of the 77 kDa nanI isoenzyme was attempted. The expressed full-length protein was found to degrade easily; a stable 50 kDa catalytic domain was therefore subcloned. This domain was overexpressed in Escherichia coli and produced crystals belonging to space group P2 1 2 1 2 1 , with unit-cell parameters a = 96.98, b = 69.41, c = 72.69 A Ê and one monomer per asymmetric unit. The crystals diffract to at least 0.92 A Ê . A molecular-replacement solution was obtained using the catalytic domain of the sialidase from the leech Macrobdella decora.

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Section: Resultsmentioning

confidence: 84%

Section: Expression Purification and Crystallization Of Nanimentioning

confidence: 96%

Section: Expression Purification and Crystallization Of Nanimentioning

confidence: 99%

Section: Subcloning Expression and Purificationmentioning

confidence: 99%

See 2 more Smart Citations

Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, fromClostridium perfringens

Newstead

Chien²,

Taylor

et al. 2004

Acta Crystallogr D Biol Cryst

Self Cite

View full text Add to dashboard Cite

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“…NanH, also called sialidase, is an extracellular neuraminidase (Trost et al 2010) that contributes to the recognition of sialic acids exposed on the surface of host cells (Kim et al 2010). In pathogenic bacteria, these enzymes are considered important virulence factors as they assist with the propagation or invasion of bacteria within the host (Sheu et al 2002). PknG protein has already been identified in species of the genus Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus, and it is said to be responsible for inhibiting the formation of phagolysosomes, consequently allowing the intracellular survival of mycobacteria (Niebisch et al 2006).…”

Section: Discussionmentioning

confidence: 99%

In silico analyses and design of a chimeric protein containing epitopes of SpaC, PknG, NanH, and SodC proteins for the control of caseous lymphadenitis

Silva

Pinho

Bezerra

et al. 2021

Appl Microbiol Biotechnol

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Caseous lymphadenitis (CLA) is an infectious disease that affects goats and sheep causing drastic impacts on milk and meat production and is caused by Corynebacterium pseudotuberculosis. The disease can be prevented through vaccination but currently, vaccines demonstrate limited efficacy consequently leading to a need for the development of new ones. Here, we described the in silico development of a new chimeric protein constructed with epitopes identified from the sequences of the genes nanH, pknG, spaC, and sodC, previously described as potential vaccinal targets against C. pseudotuberculosis. The chimera was expressed, purified, and its immunogenicity was evaluated using sera of immunized mice. Results indicate the chimeric protein was able to stimulate antibody production. Additionally, analysis using serum from naturally infected goats showed that the protein is recognized by sera from these animals, indicating the possibility for using this chimera in new diagnostic methods. Key points• The chimera was expressed with 52 kDa and a yield of 7 mg/L after purification.• The chimera was recognized by the sera of animals immunized with this formulation.• Chimera reacted with the serum of goats naturally infected with C. pseudotuberculosis.

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Sialidase Production and Genetic Diversity in Clostridium perfringens Type A Isolated from Chicken with Necrotic Enteritis in Brazil

2014

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The sialidase activity and genetic diversity of 22 Clostridium perfringens strains isolated from chickens with necrotic enteritis were determined. Sialidase activity was detected in 86.4 % of the strains. All C. perfringens showed a high value of similarity (>96 %), and they were grouped into seven clusters clearly separated from the other reference bacterial strains. From these clusters four patterns were defined in accordance with their phenotypic (sialidase production and antibiotic resistance profile) and genotypic (presence of nanI and nanJ genes) characteristics. Our results showed heterogeneity among strains, but they were genotypically similar, and it is suggested further studies are needed to better understand the pathogenesis of necrotic enteritis.

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Cloning, expression, and deletion analysis of large nanH of Clostridium perfringens ATCC 10543

Cited by 5 publications

References 19 publications

Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, fromClostridium perfringens

Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, fromClostridium perfringens

In silico analyses and design of a chimeric protein containing epitopes of SpaC, PknG, NanH, and SodC proteins for the control of caseous lymphadenitis

Sialidase Production and Genetic Diversity in Clostridium perfringens Type A Isolated from Chicken with Necrotic Enteritis in Brazil

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