Expression screening

Bussow K et al. A human cDNA library for high-throughput protein expression screening. Genomics 2000 65 1—8. 

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 ... 

SIGEX substrate-induced gene-expression screening... 

Chang, S., Bezprozvannaya, S., H, S. and Olson, E.N. (2005) An expression screen reveals modulators of class II histone deacetylase phosphorylation. Proceedings of the National Academy of Sciences of the United States of America, 102, 8120-8125. 

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). 

There are two yeast expression hosts that have an established track record for high-level production of heterologous proteins, namely Saccharomyces cerevisiae and Pichia pastoris. HTP expression screening using microplate formats has been reported for both these yeasts by Lang and coworkers (Holz et ah, 2002, 2003 Boettner et ah, 2002). In both cases standard protocols have been miniaturized with cells cultured in either 1.5 ml cultures in 96-deep-well plates for S. cerevisiae or 2 ml cultures in 24-deep-well plates for P. pastoris. Soluble... 

The most commonly used culture system for scale-up of E. coli is the shake flask. This conventional methodology has been shown to produce the quantity and quality of protein required with good scalability from plate-based expression screening. In general, cell-line, medium, growth conditions, and induction method are determined by the small-scale expression screen. Typically, the shake-flask is filled to one-quarter of its capacity to allow for adequate aeration of the culture. The generic OPPF... 

Bahia, D., Cheung, R., Buchs, M., Geisse, S. and Hunt, I. (2005). Optimisation of insect ceU growth in deep-well blocks development of a high-throughput insect cell expression screen. Protein Expr. Purif. 39, 61-70. 

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. 

Folkers, G. E., van Buuren, B. N. and Kaptein, R. (2004). Expression screening, protein purification and NMR analysis of human protein domains for structural genomics. /. Struct. Fund. Genomics 5,119-131. 

Assersohn et al. (27) reported that the median recovery of breast FNAs was 202,500 cells, which translated to between 0.81 and 1.42 p.g of total RNA, well below the typical requirement for a microarray experiment. Consequently, many researchers routinely incorporate amplification methods into their gene expression screens. There is currently no consensus in the literature with regard to the best amplification strategy to utilize. Indeed, even the fidelity of the various amplification methods available is the subject of dispute. Some researchers report that amplified RNA can substitute for total RNA in gene expression experiments (28,29,30) while others have shown that amplification of RNA introduces bias to the results (31, 32). It is quite possible, however, that this... 

Key Words Protein expression screen hexahistidine tag dot blot. 

Fig. 1. Expression screen dot blots of (A) Xylella samples and (B) Ciona samples, grown for 4 h or overnight (o/n) at 18, 25, 30, and 37°C. The standard curve is shown below. The spots outlined with squares indicate samples that would be chosen for protein purification.

The chapter on expression screening describes a simple method to quickly identify the optimal condition for expression induction. The amount of IPTG (0.1 to 1 mM), optimal temperature (18°Cto 37°C), and length of the induction time (4 hto overnight) should be identified to improve the likelihood of producing soluble protein and improve the yields of soluble proteins. Additionally, purification from several protein expression conditions can be tested in parallel. 

Anderson CN, Grant SG (2006) High throughput protein expression screening in the nervous system-needs and limitations. J Physiol 575 367-372... 

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... 

Freeman, T., High throughput gene expression screening Its emerging role in drug discovery, Med. Res. Rev. 20,197, 2000. 

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