A Novel Strategy for Oriented Protein Immobilization

A new strategy for oriented immobilization of proteins was proposed. The strategy contains two steps. The first step is to search for a docking site away from the active site on the protein surface. The second step is trying to find a ligand that is able to grasp the targeted site of the protein. To avoid ligand binding to the active site of protein, the targeted docking site is selected to own opposite charges to those near the active site. To enhance the ligand-protein binding, both hydrophobic and electrostatic interactions need to be included. The targeted docking site should therefore contain hydrophobic amino acids. The ligand is then selected through the help of molecular docking simulations. The enzyme α-amylase derived from Aspergillus oryzae (TAKA) was taken as an example for oriented immobilization. The active site of TAKA is surrounded by negatively charged amino acids. All the possible hydrophobic sites on the surface of TAKA were evaluated by the free energy estimation through benzene docking. A hydrophobic site on the opposite side of TAKA's active site was found to be positive in net charges. A possible ligand, 3,3',4,4' - Biphenyltetra- carboxylic acid (BPTA), was found to catch TAKA by the designated docking site. Then, the BPTA molecules were grafted onto silica gels and measured the affinity of TAKA adsorption and the specific activity of thereby immobilized enzymes. It was found that TAKA had a dissociation constant as low as 7.0×10 -6 M toward the ligand BPTA on silica gel. The increase in ionic strength has little effect on the adsorption of TAKA, which indicated the existence of hydrophobic interaction between ligands and proteins. The specific activity of the immobilized TAKA was compared with the randomly adsorbed TAKA on primary amine containing silica gel. It was found that the orderly immobilized TAKA owns a specific activity twice as high as the one randomly adsorbed by ionic interaction. Keywords—Protein Oriented immobilization, Molecular docking, ligand design, surface modification. the efficiency of biotransformation and to enhance the sensitivity of bioassays. Two major techniques have been adopted for oriented immobilization. One strategy was to produce recombinant proteins containing histidine residues in either amino or carboxyl end of the protein. The recombinant proteins were then immobilized through metal chelating.(3, 4) The other strategy was to immobilize proteins through bio- affinity interaction. Frequently adopted interactions include antibody-antigen, biotin-avidin, DNA-DNA, aptamer-protein, affibody-protein interactions.(5-7) However, some bio-ligands themselves suffer from denaturization and misorientation. A new strategy has been proposed for designing a synthetic affinity ligand for directed immobilization of proteins. The ligand is designed to grasp a specific spot on the protein surface far away from its active site. Both hydrophobic and electrostatic interactions are included in the protein-ligand contacts. Firstly, the amino acids and estimate the net charges near the active site of the targeted protein are examined. Secondly, molecular docking simulation of benzene molecule is performed to rank the possible hydrophobic sites on the protein surface. Then, a hydrophobic site away from the active site was selected and surrounded by amino acids of opposite charges to the active site. Taking this specific site as the target for ligand screening, molecular docking simulation is again performed to select the suitable ligands. In this study, the α- amylase derived from Aspergillus oryzae (TAKA) was selected as an example. The selected ligand was chemically attached to a silica gel support to test for adsorptive protein immobilization. The affinity of TAKA adsorption and the specific activity of the immobilized protein were measured to examine the ligand selection.