A METAL-CHELATING PLURONIC FOR IMMOBILIZATION OF HISTIDINE-TAGGED PROTEINS AT INTERFACES - IMMOBILIZATION OF FIREFLY LUCIFERASE ON POLYSTYRENE BEADS

Pluronic is a surface-active poly(ethylene oxide)-poly(propylene oxide )-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer. The PEO chain s of the triblock form a hydrophilic, protein-repelling interface at h ydrophobic surfaces. The protein-repelling property of Pluronic surfac tants was used as an activity-preserving foundation on which to develo p a scheme for specific protein immobilization at hydrophobic interfac es through immobilized metal affinity. A nitrilotriacetic acid (NTA) g roup was coupled to the terminal hydroxyl groups of the PEO chains of Pluronic F108 to create a metal-chelating Pluronic (F108-NTA). Recombi nant firefly luciferase (FFL) with C-terminal histidine tags was used as a test protein. Histidine-tagged FFL adsorbed strongly to untreated polystyrene beads but retained much less than 1% of its bioluminescen ce activity. An equivalent amount of histidine-tagged FFL bound to pol ystyrene beads treated with the chelating Pluronic, but only in the pr esence of Ni2+ ions. The firefly luciferase immobilized on the chelati ng Pluronic retained at least 93% of its bioluminescence activity. The results demonstrate that the chelating Pluronic is a simple and versa tile reagent for specific, oriented immobilization of histidine-tagged proteins on hydrophobic interfaces.