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.

• Ching-Wei Tsai
• Chih-I Liu
• Ruoh-Chyu Ruaana

• Protein Oriented immobilization
• Molecular docking
• ligand design
• surface modification.

[1] F. Rusmini, Z. Zhong, and J. Feijen, "Protein Immobilization Strategies
for Protein Biochips," Biomacromolecules, vol. 8, no. 6, pp. 1775-1789,
2007.
[2] U. Bilitewski, "Protein-sensing assay formats and devices," Analytica
Chimica Acta, vol. 568, no. 1-2, pp. 232-247, 2006.
[3] N. Haddour, S. Cosnier, and C. Gondran, "Electrogeneration of a
Poly(pyrrole)-NTA Chelator Film for a Reversible Oriented
Immobilization of Histidine-Tagged Proteins," Journal of the American
Chemical Society, vol. 127, no. 16, pp. 5752-5753, 2005.
[4] H. M. Chen, W. C. Wang, and S. H. Chen, "A Metal-Chelating
Piezoelectric Sensor Chip for Direct Detection and Oriented
Immobilization of PolyHis-Tagged Proteins," Biotechnology Progress,
vol. 20, no. 4, pp. 1237-1244, 2004.
[5] F.-F. Liu, T. Wang, X.-Y. Dong, and Y. Sun, "Rational design of affinity
peptide ligand by flexible docking simulation," Journal of
Chromatography A, vol. 1146, no. 1, pp. 41-50, 2007.
[6] G. Demirel, M. O. Caglayan, B. Garipcan, M. Duman, and E. Piskin,
"Oriented immobilization of IgG on hydroxylated Si(001) surfaces via
protein-A by a multiple-step process based on a self-assembly
approach," Journal of materials science vol. 42, pp. 9402-9408 2007.
[7] Y. Kwon, Z. Han, E. Karatan, M. Mrksich, and B. K. Kay, "Antibody
Arrays Prepared by Cutinase-Mediated Immobilization on Self-
Assembled Monolayers," Analytical Chemistry, vol. 76, no. 19, pp.
5713-5720, 2004.
[8] P. Khodade, R. Prabhu, N. Chandra, S. Raha, and R. Govindarajan,
"Parallel implementation of AutoDock," Journal of Applied
Crystallography, vol. 40, no. 3, pp. 598-599, 2007.
[9] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
J. R. Cheeseman, J. J. A. Montgomery, T. Vreven, K. N. Kudin, J. C.
Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci,
M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, and Y. H. T.
Nakajima, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P.
Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W.
Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J.
Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C.
Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B.
Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski,
B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L.
Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A.
Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M.
W. Wong, C. Gonzalez, and J. A. Pople, Gaussian, , "Gaussian 03,
Revision C.02," Inc., Wallingford CT, 2004.
[10] W. Humphrey, A. Dalke, and K. Schulten, "VMD - Visual Molecular
Dynamics," J. Molec. Graphics, vol. 14, pp. 33-38, 1996.
[11] K. Lorentz, "Approved Recommendation on IFCC Methods for the
Measurement of Catalytic Concentration of Enzymes Part 9. IFCC
Method for a-Amylase (1,4- a -D-Glucan 4-Glucanohydrolase, EC
3.2.1.1)," Clinical Chemistry and Laboratory Medicine, vol. 36, no. 3,
pp. 185-203, 1998.
[12] Y. Matsuura, M. Kusunoki, W. Harada, and M. Kakudo, "Structure and
Possible Catalytic Residues of Taka-Amylase A," J Biochem, vol. 95,
no. 3, pp. 697-702, January 1, 1984, 1984.