CDNA, fractionation

Multiple lines of evidence suggest that SPF, a single protein within the 20-25 kDa pheromone fraction, is responsible for female behavioural response. First, this major protein component within the D. ocoee fraction was genetically very similar to the precursor of sodefrin (Palmer, Watts, Houck, Picard and Arnold 2007), and sodefrin is a known reproductive pheromone in newts (Kikuyama, Toyoda, Ohmiya, Matsuda, Tanaka and Hayashi 1995 Kikuyama and Toyoda 1999). Second, a separate study showed that the cDNA library of proteins expressed in male D. ocoee mental glands contained a high proportion (25%) of... 

The recovered RNA is reverse transcribed to cDNA and amplified. The next-generation RNA pool, generated again by in vitro transcription, is already enriched in RNA molecules that bind to their target. The procedure is repeated with rising stringency until the random RNA pool is purified to a fraction of RNA... 

Okazaki N, Kikimo R, Ohara R, et al. (2002) Prediction of the coding sequences of mouse homologues of KIAA gene I. The complete nucleotide sequences of 100 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries DNA Res 9, 179-188. 

Cell or tissue lysates represent the crudest tissue fraction. Use of lysates is practical when the level of enzyme is relatively high. However, lysates have a tendency to form aggregates, which limits the maximum protein concentration which can be used and also limits the time for which the reaction to be studied is linear. Typically, protein concentrations of less than 3 mg/ml must be used and incubation time is limited to about 30 min. This can be an important limitation for substrates which are metabolized very slowly. In addition, cell fractionation is not limited to primary tissues. Fractions can be prepared from cDNA-expressing cell lines. This can provide a level of convenience to the researcher relative to the demands of maintaining multiple cell lines for extended periods of time. 

Generation of potential human metabolites for structural identification prior to administration of the drug candidate to humans can be performed with either cDNA-expressed enzymes or tissue fractions. This allows identification of potential human metabolites and development of appropriate analytical methods prior to clinical trials. Generation and identification of pharmacologically active human metabolites early in the development process can be beneficial for obtaining appropriate patent protection. 

A functional uricase was obtained by expression in E. coli of a soybean N-35 cDNA driven by the bacterial lacZ promoter (Suzuki Verma, 1991). The uricase activity was mainly found in the cytoplasmic fraction of E. coli and had the same pH optimum and apparent Km values as in the nodules. That N-35 is able to assemble into a functional, tetrameric holoenzyme in E. coli indicates that post-translational modifications, or the presence of peroxisomes, is not essential for its proper assembly and function in this organism. However, N-35 is not active and does not accumulate to any significant levels when it is expressed in transgenic tobacco under the control of the CaMV-35S promoter (our unpublished data). 

Fig. 6. (Opposite page) A high-throughput production method for screening proteins from cDNA libraries. Authentic (A) and glutathione -transferase (GST)-fused (G) proteins in the reaction mixtures after a semi-automated polymerase chain reaction/ transcription and translation from 54 different cDNAs separated by sodium dodecyl sul-fide-polyaciylamide gel electrophoresis and stained with Coomassie Brilliant Blue. T and S, respectively, mark total translation product and the supernatant fraction after centrifugation at 30,000g for 15 min.

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