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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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Lectin-binding activity of the bifunctional hybrid CmAP/MBL-AJ mutants. The lectin-bound complexes with mucin (axis X) were monitored by measuring the phosphatase activity of CmAP/MBL-AJ (axis Y).
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pone-0112729-g013: Lectin-binding activity of the bifunctional hybrid CmAP/MBL-AJ mutants. The lectin-bound complexes with mucin (axis X) were monitored by measuring the phosphatase activity of CmAP/MBL-AJ (axis Y).

Mentions: For the experimental testing of the lectin-binding activity, W100A, H103Q, A156N, F159K and A156N/F159K mutants were expressed in E.coli. The chimeric mutants of CmAP/MBL-AJ remained the lectin activity in the range of the increasing of the level of affinity W100A– H103Q– A156N– F159K to mucin that was in accord with the calculated data (Table 2, Fig. 13). The W100A, Q103H, A156N mutations decreased the lectin-binding activity in comparison with the wild-type CmAP/MBL-AJ by 14±3%, 12±2% and 8±2%, respectively.


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)

Lectin-binding activity of the bifunctional hybrid CmAP/MBL-AJ mutants. The lectin-bound complexes with mucin (axis X) were monitored by measuring the phosphatase activity of CmAP/MBL-AJ (axis Y).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112729-g013: Lectin-binding activity of the bifunctional hybrid CmAP/MBL-AJ mutants. The lectin-bound complexes with mucin (axis X) were monitored by measuring the phosphatase activity of CmAP/MBL-AJ (axis Y).
Mentions: For the experimental testing of the lectin-binding activity, W100A, H103Q, A156N, F159K and A156N/F159K mutants were expressed in E.coli. The chimeric mutants of CmAP/MBL-AJ remained the lectin activity in the range of the increasing of the level of affinity W100A– H103Q– A156N– F159K to mucin that was in accord with the calculated data (Table 2, Fig. 13). The W100A, Q103H, A156N mutations decreased the lectin-binding activity in comparison with the wild-type CmAP/MBL-AJ by 14±3%, 12±2% and 8±2%, respectively.

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