Synthesis

Solid-Phase Combinatorial Synthesis of a Ligand Library (Fig. 3) 1. Epoxyactivation of agarose beads. The required amount of Sepharose CL-6B is washed with 40 mL of distilled water g of gel on a sinter funnel (see Note 7). The washed agarose is transferred to a 1-L conical flask, and 1 mL of distilled water g of gel is added. To this moist gel, 0.8 mL of 1 MNaOH mL of gel and 1 mL of epi-chlorohydrin mL of gel are added. The slurry is incubated for 10-12 h at 30 C on Fig. 2. Comparison...

Introduction

Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines (1). Escherichia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility, and toxicity in this environment, resulting in differing rates of protein degradation, From Methods...

Screening

Screening of affinity ligands is done by affinity chromatography (performed at room temperature) (Fig. 4). 1. The affinity ligands (1 g of moist gel) are packed into 4-mL columns (0.8 o 6 cm). Each matrix is washed with 2 o 3 mL regeneration buffer and then with distilled water to bring the pH to neutral. The resins are equilibrated with 10 mL of equilibration buffer. 2. Protein to be tested is reconstituted to 1 mg mL in equilibration buffer and the absorbance at 280 nm measured. Protein...

Materials

Various software tools are available with which to build, handle, and investigate structures in silico, and there are also quite a few docking programs available. All have their own set of features, strengths, and weaknesses. It is impossible to describe a generic protocol for the performance of docking experiments using any combination of software modules. Fortunately, the general setup will be similar for alternative programs. Therefore, the following subsections describe the use of a...

Miscellaneous

Putative Active Site with Spheres (PASS) AGENT2.0 http www.pharma.ethz.ch pc Agent2 GRID www.moldiscovery.com docs grid21 References 1. Hou, T. J. and Xu, X. J. (2004) Recent development and application of virtual screening in drug discovery an overview. Curr. Pharm. Des. 10, 1011-1033. 2. Taylor, R. D., Jewsbury, P. J., and Essex, J. W. (2002) A review of protein-small molecule docking methods. J. Comput. Aided Mol. Des. 16, 151-166. 3. Bohm, H. J. and Stahl, M. (2002) The use of scoring...

Optimization of Expression Level for Assays

Before screening, it is necessary to determine the optimal amount of adenovirus to use for each assay and for each cell line. If the goal is to identify small molecules that increase expression of the reporter protein, it may be optimal to adjust adenovirus concentrations such that the reporter protein is expressed at low levels in the absence of screening compounds. On the other hand, if the goal is to identify compounds that suppress expression of the reporter protein, it may be preferable to...

Notes

Solubility of the agarose and ketone-modified agarose is limited. To dissolve agarose in 20 DMSO acetate buffer (pH 4.5), the solution needs to be heated to 50 C and shaken gently overnight. 2. Microarrays prepared from macromolecular scaffold conjugates were found to be stable in all washing procedures used in our laboratory. However, mechanical wiping must be avoided, as it will damage the microarrays. 3. For the cell-binding arrays, 300- im spots are preferred 100- im spots can be used for...

Final Remarks

The exhaustive body of literature on virtual screening, docking, and scoring tools renders it impossible to describe every available program and method in detail. New docking algorithms and scoring functions are developed continually. Validation studies using various docking and scoring tools appear on a regular basis, enabling a more thorough comparison of the different methods. In this chapter, we have tried to give a practical guide to setting up a docking run, without going into too much...

Filename MOL2 File MLTmol2

Running the GOLD Docking Program GOLD comes with its own graphic user interface (GUI), allowing the program to run interactively. The user can choose among various running modes, each with its own demand of central processing unit (CPU) time and accuracy (Fig. 5). A configuration file specifying the necessary input parameters can be set up through the GUI (Fig. 6) and either saved or run interactively (however, see Note 1). The generated configuration file (Fig. 7) can be edited manually...

Construction of the Expression Plasmid for Full Length p53

The E. coli expression system used is based on the pQE-80L expression system. Sequences inserted into the multiple cloning site can be expressed as native proteins bearing an N-terminal hexahistidine tag upon IPTG induction of the T5 promoter in suitably transformed E. coli cells. 3.1.2. Amplification and Cloning of the Wild-Type p53 Gene As a Fusion to BCCP All DNA manipulations were carried out using standard recombinant DNA methods (18) to construct the expression plasmids, and are...

Aldo Jongejan Chris de Graaf Nico P E Vermeulen Rob Leurs and Iwan J P de Esch

In silico docking techniques are being used to investigate the complementarity at the molecular level of a ligand and a protein target. As such, docking studies can be used to identify the structural features that are important for binding and for in silico screening efforts in which suitable binding partners can be identified. Here we describe a practical approach for setting up docking simulations using different docking programs. We also cover the analysis and rescoring of the obtained...

Batch Mode Cell Free Translation

This protocol is for the 50 pL batch-type translation as reported elsewhere (11). Scale-up can be achieved if desired. 1. Prepare 50 pL of translational reaction mixture by combining 10 pL of wheat embryo extract (thus 20 ), 2 pL of 1 mg mL creatine kinase, 10 pL of 5X WEB, and 28 pL of dissolved mRNA from 10 pL transcription mixture. If necessary, add (14C)leucine (final 2 pCi mL). 2. Incubate at 26 C for 4 h (for results, see Fig. 3 in Chapter 11).

Introduction of Glyoxylyl Functionality

Several methods for introduction of glyoxylyl functionality have been developed (see Fig. 2). The first method starts with coupling of 1,3-dioxolane-2-car-boxylic acid to the amino-slide, followed by acidic hydrolysis of the dioxolane ring to yield an aldehyde moiety (19). The second method involves coupling of Boc tBu-protected serine Boc-Ser(tBu)-OH to the amino-slide, followed by trifluoroacetic acid (TFA)-mediated removal of Boc tBu groups and oxidation with sodium periodate (19). The...

Of Biomimetic Ligands for Affinity Chromatography

Roque, Geeta Gupta, and Christopher R. Lowe Affinity chromatography is ideally suited to the purification of pharmaceutical proteins due to its unique bio-specificity characteristics. Tailor-made affinity ligands that represent a promising class of synthetic affinity ligands have been developed to target specific proteins and designed to mimic peptidal templates, natural biological recognition motifs, or complementary surface-exposed residues. These biomimetic ligands have been...

Options FlexX Browse results

A window is opened in which the jobname (used earlier to submit the run) can be selected and the ligands with their corresponding FlexX score will be displayed. The Show Failed Ligand button gives ligands for which no docking solution has been obtained. The successfully docked ligands can be sorted according to their binding energy score by clicking the Sorted icon. 3.4.2. Analyzing the Results Generated by Gold The docking poses found by GOLD are stored in MOL2 format. The ligand conformations...

And Robust Extract From Wheat Embryos

One of the most convenient and promising eukaryotic cell-free translation systems is based on wheat embryos, which store all the necessary components of translation. However, conventional wheat-germ extracts are plagued with a short half-life, and consequently, with low expression of proteins. In recent findings, we proposed that the possible cause for instability is the presence in the original endosperm extract of RNA N-glycosidase, tritin, and possibly other inhibitors of translation, such...