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A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

Balabanova L, Golotin V, Kovalchuk S, Bulgakov A, Likhatskaya G, Son O, Rasskazov V - PLoS ONE (2014)

Bottom Line: Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ.The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other.The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Marine Biochemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.

ABSTRACT
A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

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The homology model of CmAP monomer with binding sites for two Zn2+ and one Mg2+ ions.
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pone-0112729-g006: The homology model of CmAP monomer with binding sites for two Zn2+ and one Mg2+ ions.

Mentions: Amino acid sequences of CmAP, HaALLP and VAP have 75% and 69% of identity and 86% and 82% of similarity, respectively. The 3D-structure of cold-active alkaline phosphatase CmAP from marine bacterium C. marina (Uniprot Q1W622) was modeled using as a template the crystal structure of the cold-active Vibrio sp. G15-21 alkaline phosphatase (Fig. 6).


A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

Balabanova L, Golotin V, Kovalchuk S, Bulgakov A, Likhatskaya G, Son O, Rasskazov V - PLoS ONE (2014)

The homology model of CmAP monomer with binding sites for two Zn2+ and one Mg2+ ions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232515&req=5

pone-0112729-g006: The homology model of CmAP monomer with binding sites for two Zn2+ and one Mg2+ ions.
Mentions: Amino acid sequences of CmAP, HaALLP and VAP have 75% and 69% of identity and 86% and 82% of similarity, respectively. The 3D-structure of cold-active alkaline phosphatase CmAP from marine bacterium C. marina (Uniprot Q1W622) was modeled using as a template the crystal structure of the cold-active Vibrio sp. G15-21 alkaline phosphatase (Fig. 6).

Bottom Line: Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ.The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other.The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Marine Biochemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.

ABSTRACT
A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

Show MeSH
Related in: MedlinePlus