Open reading frame, effects

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Bryant and DeLuca (5) purified and characterized a Type I nitroreductase from Enterobacter cloacae. The protein is a monomer of approximately 27 kDa, it has a loosely bound FMN cofactor and uses NAD(P)H as an electron donor. The substrate range of the enzyme includes nitrofurans, nitrobenzenes, nitrotoluenes and quinones. The enzyme appears to produce the hydroxylamino derivative of nitroflirazone under aerobic conditions, but the product was not thoroughly characterized. Under anaerobic conditions the product is the amine. The authors did not test the effect of metals on enzyme activity, and no inhibition studies were reported. Therefore, it is not known if this enzyme is a metalloprotein. The gene encoding the Enterobacter reductase was cloned and sequenced (7). The authors found a 651 base pair open reading frame corresponding to a protein of 23.9 kDa. Comparison of the Enterobacter and Salmonella amino acid sequences revealed 88% sequence identity between the two proteins (7). 

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