Expression libraries, cDNA

Peptide libraries Intermediaries for non peptide drugs Mirror image phage display Peptides with effector functions Select binders on unknown antigens From 2D gels From expression cDNA libraries... 

To identify relevant IgE binding components from natural source extracts, various techniques like IgE immunoscreening of expression cDNA libraries, reverse transcriptase polymerase chain reaction (PCR), or phage-display technology combined with immunoscreening are used (Wallner et al. 2004). 

In the field of proteomics, arrays can also be used to identify possible interaction partners. Here, the array is spotted with recombinant proteins or antibodies and then hybridised with labelled cell lysate or an expressed cDNA library. Additionally, techniques like two-dimensional gel electrophoresis, the identification of isolated proteins by mass spectrometry or the two-hybrid analysis are valuable tools to identify new proteins. These methods allow a large-scale study of viral and cellular proteins without knowledge of the DNA sequence (Fig. 1, IV. For review, see Pandey and Mann 2000). 

Efficient extraction of high quality RNA from a variety of plant tissues is an important first step in many procedures, such as analysis of gene expression, cDNA library construction, and in vitro translation. This procedure, which is essentially as described in Draper et al. (1), involves grinding and phenol extraction of plant material followed by differential precipitation of RNA with sodium acetate. The protocol has been successful with leaf material and cultured cells from a large number of species, however, slight adjustments may be necessary to optimize extraction from other tissues. 

Recently, a cDNA encoding a GM3-specific a2-8 SAT (SAT-2 or GD3 synthase) has been isolated from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 (Sasaki et al., 1994). Anti-GD3 antibody is used for fluorescence-activated... 

Homology screening. Using oligonucleotide probes based on known receptor sequences, search cDNA libraries for homologous sequences which may code for related receptors. The clones are then isolated and sequenced and used in expression studies to confirm the identity of the receptor. 

Crameri, R., Jaussi, R., Menz, G., and Blaser, K. (1994). Display of expression products of cDNA libraries on phage surfaces. Eur. J. Biochem. 226, 53-58. 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

Tc-SOD-1) has been cloned and expressed (Matzilevich et al., 1999, unpublished results). While antibodies to this protein recognize a dimer of 20/22 kDa in larval extracts, evidence has yet to be obtained that this gene product is represented in larval TES. In addition, several more SOD genes have been identified by an EST project from a cDNA library from this stage. It seems likely, but has yet to be demonstrated, that SODs represent an important part of the parasite s defence mechanism against immune attack, as has been shown for other nematodes (James, 1994 Ou et al, 1995). 

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