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

PLoS One. 2014 Nov 14;9(11):e112729. doi: 10.1371/journal.pone.0112729. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / chemistry
  • Alkaline Phosphatase / genetics*
  • Amino Acid Substitution / genetics
  • Animals
  • Chromatography, Gel
  • DNA Primers / genetics
  • Dimerization
  • Escherichia coli
  • Halomonadaceae / enzymology*
  • Mannose-Binding Lectin / chemistry
  • Mannose-Binding Lectin / genetics*
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Plasmids / genetics
  • Protein Conformation
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Sea Cucumbers / genetics*

Substances

  • DNA Primers
  • Mannose-Binding Lectin
  • Recombinant Fusion Proteins
  • Alkaline Phosphatase

Grants and funding

The work was supported by grant n. 12-04-00825-a from Russian Found of Fundamental Researches (http://www.rfbr.ru/rffi/ru), the Russian projects “Far Eastern” n. 13-HTΠI-12, 12-I-Π6-10, and the project “Scientific Found” of Far Eastern Federal University n. 14-08-01-11-u (http://www.dvfu.ru/en/web/fefu/). The funders supported the data collection and analysis, and decision to publish.