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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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Optimization of pET40CmAP/MBL-AJ expression: black squares - specific AP activity; red circles - total activity; blue triangles - total protein.
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pone-0112729-g002: Optimization of pET40CmAP/MBL-AJ expression: black squares - specific AP activity; red circles - total activity; blue triangles - total protein.

Mentions: The optimal conditions for the CmAP/MBL-AJ expression were the 0.2 mM concentration of IPTG and the strain cultivation at 16°C for 12 h (Fig. 2). At the final step of purification, SDS-PAGE showed a single band of the protein with a molecular mass of approximately 100 kDa, corresponding to the calculated molecular mass of the hybrid protein CmAP/MBL-AJ consisted of the mature proteins of CmAP (55 kDa), MBL-AJ (17 kDa), peptide linker (G4S)3, the plasmid fusion protein DbsC (32.5 kDa) and N- and C-terminal 6 X His tags (Fig. 1, 3). The relative molecular size determined by gel filtration corresponded to 200 kDa, suggesting a homodimeric form of the enzymatically active hybrid CmAP/MBL-AJ. CmAP/MBL-AJ appeared as tetramer at the highly alkaline pH≥9 (data not shown).


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)

Optimization of pET40CmAP/MBL-AJ expression: black squares - specific AP activity; red circles - total activity; blue triangles - total protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112729-g002: Optimization of pET40CmAP/MBL-AJ expression: black squares - specific AP activity; red circles - total activity; blue triangles - total protein.
Mentions: The optimal conditions for the CmAP/MBL-AJ expression were the 0.2 mM concentration of IPTG and the strain cultivation at 16°C for 12 h (Fig. 2). At the final step of purification, SDS-PAGE showed a single band of the protein with a molecular mass of approximately 100 kDa, corresponding to the calculated molecular mass of the hybrid protein CmAP/MBL-AJ consisted of the mature proteins of CmAP (55 kDa), MBL-AJ (17 kDa), peptide linker (G4S)3, the plasmid fusion protein DbsC (32.5 kDa) and N- and C-terminal 6 X His tags (Fig. 1, 3). The relative molecular size determined by gel filtration corresponded to 200 kDa, suggesting a homodimeric form of the enzymatically active hybrid CmAP/MBL-AJ. CmAP/MBL-AJ appeared as tetramer at the highly alkaline pH≥9 (data not shown).

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