Cell-free screening

Cell-Free Screening on Isolated Vascular Ligands or Receptors... 

Branch AD. Antisense drug discovery can cell-free screens speed the process Antisense Nucleic Acid Drug Dev 1 998 8 249-254. 

Sawasaki, T., Hasegawa, Y., Tsuchimochi, M. etal. (2002) A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Letters, 514 (1), 102-105. 

Recently it was found that obelin mRNA can be a useful tool for evaluating the efficiency of cell-free translation and for screening of translation inhibitors. 

The involvement of several tyrosine kinases in various cancers requires efficient screening methodologies for the inhibitory compounds. Screening is divided into three steps (1) primary screening against the pure isolated PTK in a cell-free system. The objective is always an ELISA format. The compounds are screened against a battery of PTKs and Ser/Ther kinases in order that the pattern of selectivity can be established quickly [2]. 

Numerous testing systems and protocols have been used to study 5-LO inhibitors in different laboratories, complicating attempts to compare compounds and series. In vitro, a variety of both cell-free and cellular preparations have been employed as primary screens. The most commonly used cell-free system is the crude cytosolic fraction from broken RBL-1 cells [25] various broken neutrophil preparations are also used, and more recently purified enzymes have occasionally been employed. The formation of 5-LO products is generally determined by radioimmunoassay or (in older work) HPLC or bioassay methods. 

A set of Saccharomyces cerevisiae reductases was screened in collaboration with J. D. Stewart s group (University of Florida). Itwas demonstrated that diketo ester la is accepted as substrate by at least three different NADP(H)-dependent reductases of this microorganism. Application of a cell-free system in preparative batches using enzyme-coupled coenzyme regeneration afforded (R)-2a with more than 99% enantiomeric excess [13]. 

Figure 6.9 Retest results of the prima hit clones from CIAA stability (13-2H to 19-10A) and productivity screening (1-4Ato 10-4C). Comparable amounts of DERA cell-free...

The CFCF described by Spirin (30) is efficient for protein production using the efficient wheat germ cell-free system, as shown in the previous section. However, this system may not be suitable for massive screening of cDNA libraries for structural genomics and production of gene products. Because the CFCF apparatus is equipped with a semipermeable membrane in the reaction chamber, the system is quite complicated to manipulate. The delicate nature of the membrane can pose problems for automating the translation process. Also,... 

Morita, E. H., Sawasaki, T., Tanaka, R., Endo, Y., and Kohno, T. (2003) A wheat germ cell-free system is a novel way to screen protein folding and function. Protein Sci. 12,1216-1221. 

Target enzymes expressed biotechnologically are also introduced to HTS systems. Merck researchers reported a new approach to drug screening by combinatorial enzymology [121], They have engineered a cell-free bacterial cell wall... 

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