Expression, purification and characterisation of chondroitinase AC II with glyceraldehyde-3-phosphate dehydrogenase tag and chaperone (GroEs-GroEL) from Arthrobacter sp. CS01

Fang, Yangtao; Fu, Xiaodan; Xie, Wancui; Li, Li; Liu, Zhemin; Zhu, Chunshen; Mou, Haijin

doi:10.1016/j.ijbiomac.2019.02.056

Cited by 6 publications

(4 citation statements)

References 24 publications

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“…In another example, the active soluble form of ADH protein was obtained through co-expression with the chaperone protein using the pG-KJE8 vector [ 39 ]. By using the chaperone GroEs-GroEL as partners, the enzyme activity of ChSase AC II increased from 3.12 U/mL to 9.15 U/mL [ 40 ]. The production of astaxanthin was further increased to 26 mg/L and 6.17 mg/g DCW (dry cell weight) by utilizing the molecular chaperone genes groES-groEL [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Improvement of Carotenoids’ Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies

Zhou,

Huang,

Yang

et al. 2024

Microorganisms

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Canthaxanthin is an important antioxidant with wide application prospects, and β-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of β-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of β-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of β-carotene ketolase in Escherichia coli. In particular, the production of soluble β-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from β-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of β-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different β-carotene ketolase. This study not only laid a foundation for further research on the improvement of β-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production.

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Section: Discussionmentioning

confidence: 99%

Improvement of Carotenoids’ Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies

Zhou,

Huang,

Yang

et al. 2024

Microorganisms

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“…The elution fraction was desalted by dialysis with 50 mM Tris-HCl buffer (pH 7.5) as the mobile phase. The Mw of YsHyl8A was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [20,21]. The protein concentration was determined using the method of Lowry and colleagues using bovine serum albumin (BSA) as the standard [22].…”

Section: Acquisition Of Recombinant Gene and Expression And Purificat...mentioning

confidence: 99%

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298

Zhang

Han

et al. 2022

Molecules

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A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0–11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases.

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“…The human CRISP1 constructs that were transformed into different E. coli strains are summarized in Supplementary Figure 2A. The E. coli pG-KJE8/ BL21 strain possesses two chaperone systems that enhance the solubility of proteins [29][30][31] and the Origami 2(DE3) pLysS strain improves disulfide bond formation in the cytoplasm of E. coli 32 . Initially, the human CRISP1 coding sequence was optimized according to the codon bias of E.coli 33 and fused with the N-terminal His-tag.…”

Section: Reagentsmentioning

confidence: 99%

The less conserved metal-binding site in human CRISP1 remains sensitive to zinc ions to permit protein oligomerization

Sheng

Gadella

Olrichs

et al. 2021

Sci Rep

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Cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and PR-1 (CAP) superfamily that is characterized by the presence of a conserved CAP domain. Two conserved histidines in the CAP domain are proposed to function as a Zn2+-binding site with unknown function. Human CRISP1 is, however, one of the few family members that lack one of these characteristic histidine residues. The Zn2+-dependent oligomerization properties of human CRISP1 were investigated using a maltose-binding protein (MBP)-tagging approach in combination with low expression levels in XL-1 Blue bacteria. Moderate yields of soluble recombinant MBP-tagged human CRISP1 (MBP-CRISP1) and the MBP-tagged CAP domain of CRISP1 (MBP-CRISP1ΔC) were obtained. Zn2+ specifically induced oligomerization of both MBP-CRISP1 and MBP-CRISP1ΔC in vitro. The conserved His142 in the CAP domain was essential for this Zn2+ dependent oligomerization process, confirming a role of the CAP metal-binding site in the interaction with Zn2+. Furthermore, MBP-CRISP1 and MBP-CRISP1ΔC oligomers dissociated into monomers upon Zn2+ removal by EDTA. Condensation of proteins is characteristic for maturing sperm in the epididymis and this process was previously found to be Zn2+-dependent. The Zn2+-induced oligomerization of human recombinant CRISP1 may shed novel insights into the formation of functional protein complexes involved in mammalian fertilization.

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Expression, purification and characterisation of chondroitinase AC II with glyceraldehyde-3-phosphate dehydrogenase tag and chaperone (GroEs-GroEL) from Arthrobacter sp. CS01

Cited by 6 publications

References 24 publications

Improvement of Carotenoids’ Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies

Improvement of Carotenoids’ Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298

The less conserved metal-binding site in human CRISP1 remains sensitive to zinc ions to permit protein oligomerization

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