Expression and functional characterization of recombinant tributyltin-binding protein type 2

Area of Science:

• Biochemistry
• Environmental Toxicology
• Molecular Biology

Background:

• Tributyltin-binding proteins (TBT-bps) are fish lipocalins involved in binding and detoxifying tributyltin (TBT).
• Understanding TBT-bp function is crucial for assessing TBT's environmental impact on aquatic organisms.

Purpose of the Study:

• To produce and characterize recombinant TBT-bp2 (rTBT-bp2) using a baculovirus-silkworm expression system.
• To compare the TBT-binding properties of rTBT-bp2 with previously characterized rTBT-bp1.

Main Methods:

• Constructed a baculovirus-silkworm expression system for TBT-bp2.
• Produced recombinant TBT-bp2 (rTBT-bp2) in silkworm hemolymph.
• Investigated ligand binding using DAUDA and TBT in competition assays.

Main Results:

• rTBT-bp2 bound to DAUDA with a dissociation constant of 0.97 µM, indicating conserved lipocalin structure.
• rTBT-bp2 exhibited strong binding affinity to TBT (inhibition constant of 0.29 µM) in its central ligand pocket.
• Negative cooperativity was observed in rTBT-bp2 binding, suggesting multiple binding sites.

Conclusions:

• The study successfully produced functional rTBT-bp2, confirming conserved lipocalin structure and TBT-binding capability.
• rTBT-bp2 plays a role in TBT binding, potentially contributing to detoxification mechanisms in fish.
• Further research is needed to elucidate the specific endogenous functions of TBT-bps in response to xenobiotics.