Cloning expression screening

Other methods include detection of production of mRNA or protein in response to the presence of the substrate (substrate-induced gene-expression screening). The success of this method is dependent on the retention of native upstream regulatory regions of genes in the clones which can switch on production of mRNA or enzyme in response to the stimulus of the presence of substrate. The method is used in screening libraries derived by random ... 

In general, protein target identification often employs genetic techniques such as expression cloning, expression profiling, screening of yeast mutations, and yeast three-hybrid assays. None of these techniques works for every situation. 

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

Berrow, N. S., Alderton, D., Sainsbury, S., Nettleship, J., Assenberg, R., Rahman, N., Stuart, D. 1. and Owens, R. J. (2007). A versatile Ugation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, E45,1-12. 

The dimension of an antibody library, typically defined as the number of clones bearing a suitable selectable marker (antibiotic resistance) and, containing the full-size antibody gene (detectable by PCR screening 32), does not necessarily correspond to the functional dimension of a library, which requires that the clones express properly folded antibodies. An approximation for the determination of the functional dimension of a library consists of determining what percentage of clones expresses antibodies, for example, using immunoblot techniques. 

Screen, S. E., and St. Leger, R. J. (2000). Cloning, expression, and substrate specificity of a fungal chymotrypsin. Journal of Biological Chemistry, 275, 6689-6694. 

Both groups used expression cloning to screen cDNA libraries obtained from NG 108-15 mouse neuroblastoma-rat glioma hybridoma cells. These cells were a logical choice for delta opioid receptor cloning efforts, since they express high density of delta receptors and can be produced in great quantities in cell culture. 

Augenlicht LH, Kobrin D (1982) Cloning and screening of sequences expressed in a mouse colon tumor. Cancer Res 42(3) 1088-1093... 

With access to clones for one receptor type, it has been possible in several cases to isolate clones rapidly for other receptor types using cross-hybridization as exemplified by opioid receptors and somatostatin receptors. However, clones for the abundant Y2 receptor have resisted detection with Y1 probes. Instead, three different laboratories have recently succeeded by expression screening to isolate Y2 clones using PYY as... 

The first gene encoding a reverse gyrase, TopR, was cloned by screening a Agt 11 expression library of Sulfolobus acidocaldarius genomic DNA, with polyclonal... 

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