A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.     

Serogenetic variation in four caste populations of Haryana, India

The phenotypes and gene frequencies of 3 blood groups, 7 red-cell enzymes and a serum protein were studied in 4 caste population groups of Haryana, North India. The results indicate that the distribution of these blood markers is rather homogeneous in the 4 groups and generally resembles that observed in various populations from neighbouring North Indian states.     

Studies on triacylglycerol ester hydrolase from bat adipose tissue

Triacylglycerol ester hydrolase was isolated from bat adipose tissue and characterized. The partially purified enzyme had pH optimum of 8.6 and a K_m value of 0.6 mM. The enzyme was denaturated upon freezing and thawing, which was prevented by 25% glycerol. The enzyme was activated by EDTA and NaCl, while it was inhibited by serum and bovine serum albumin. Heparin, sodium fluoride and diisopropyl fluorophosphate had no effect on triacylglycerol ester hydrolase activity. It hydrolyzed triglycerides partially. Triacylglycerol ester hydrolase lost its activity during delipidation but it was reactivated by endogenous lipids and phospholipids, viz. phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin. The enzyme shows kinetic properties altogether different from lipoprotein lipase and hormone sensitive lipase     

First structural evidence for the mode of diffusion of aromatic ligands and ligand-induced closure of the hydrophobic channel in heme peroxidases

The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole (amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules, including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H₂O₂ to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic channel as well as the binding of H₂O₂. This explains the amitrole toxicity. From binding studies, the dissociation constant (K _d) for amitrole with lactoperoxidase was found to be approximately 5.5 × 10⁻⁷ M, indicating high affinity.     

A geometry optimization and molecular electrostatic potential mapping study of structure-activity relationship for some anti-Alzheimer agents.

Molecular geometries of some substituted (pyrroloamino)pyridines which possess anti-Alzheimer activity were optimized and potential-derived CHelpG point charges were computed using ab initio SCF molecular orbital approach employing the 3-21G basis set. AM1 molecular orbital calculations were performed using these optimized geometries and thus optimized Hybridization. Displacement Charges (HDC) combined with L?wdin charges continuously distributed in three dimension were obtained. Molecular electrostatic potential (MEP) maps of the molecules were obtained in two ways: (i) using the HDC-based model with the help of which MEP minima near the molecules were located, and (ii) using the CHelpG point charges, MEP values on the van der Waals surfaces of the molecules were computed. The MEP maps computed using both the methods have negative MEP regions near the pyridine nitrogen atom which appears to be the main binding site of the molecules with the appropriate receptor. Both electrostatic interaction and lipophilic association between these molecules and the receptor appear to contribute to biological activity.     

Adenovirus vaccine strains genetically engineered to express HIV-1 or HBV antigens for use as live recombinant vaccines

Types 4 and 7 adenovirus are currently used as live, oral vaccines for the prevention of adenovirus respiratory disease in military recruits. These vaccine strains have been genetically engineered in order to express HIV-1 or HBV antigens in infected cells. A dog model was developed to evaluate the immunogenicity of these recombinant vaccines. Dogs inoculated with live adenovirus-HBV recombinant vaccine produced antibody against hepatitis B surface antigen.