A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

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.

Genetically modified proteins: functional improvement and chimeragenesis

“…68 The same effect was observed in the double mutant of the recombinant hybrid mannan-binding holothurian lectin CmAP/MBL-AJ (Table 1). 44 It has been experimentally confirmed that the double mutation A137N/F159K has synergy effect on the lectin-binding activity of CmAP/MBL-AJ enhancing its activity by 25±5%, 40±2% and 28±3% in comparison with the wild type lectin and the single mutants A137N and F159K, respectively. In silico mutagenesis calculations and molecular docking with the model oligosaccharide have revealed that the double mutation A137N/F159K lead to the significant rearrangement of the amino acid residues around the lectin binding site, where the residue Leu in the mutant position F159K becomes able to react with the ligand forming the additional two ionic and one hydrogen bonds (Fig.…”

“…The synthesis of hybrid proteins by domain swapping or the ligation of chemically or biologically synthesized peptides are often used for biological probing. 41,42,43,44 Combinatorial (random insertion, circular permutation, homologous and nonhomologous recombination) and non-combinatorial (rational) methods of chimeragenesis based on the original DNA shuffling or restriction-enzyme-based shuffling are also useful for the study of molecular structure/function at the specific subunit and/or domain levels. 39,40,45 Directed evolution of DNA is one of the most important advances in biology today.…”

“…23 Molecular modeling, docking with substrate/inhibitor, dynamic simulation can facilitate adoption of decision about the protein structure from a set of the mutants that should be realized through recombinant production to obtain the desired properties. 26,29,44,46,47 Based on the information-driven combinatorial design, the directed evolution experiments consisted of mutation, recombination and screening processes identify interesting mutants. In vitro compartmentalization (IVC) is an in vitro gene screening system for directed evolution of proteins.…”

“…The synthesis of hybrid proteins by domain fusion or the ligation of chemically or biologically synthesized peptides improves apparently a functional unit utility and is generally used for biological probing approach such as biosensors and bioassays, molecular imaging, targeted cancer or antiinflammatory therapy, regulated drug delivery. 41,42,43,44,78,79,80,81 Hybrid proteins may possess two or more functional activities realized by different modes of action indicating that the molecules of hybrid act as several distinct pharmacophores. Many pharmaceutically or industrially relevant enzymatic processes can also utilize hybrid enzymes with multiple domains.…”

“…It improves the recombinant protein's properties such as foldability, solubility and functionality. 44,82,86 The decision of whose domain should follow downstream of C-terminal end depends on the many factors, particularly the possibility of quick folding during the recombinant production or the involvement of the N-or C-terminal ends in the active site formation or oligomerization.…”