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Translate High Yields of Biologically Active Proteins

ce. Figure 1 lists the genes translated, the corresponding protein molecular weights, and the protein yields. While yields varied with the translated protein, in all but one case, 1030 g of protein were produced per reaction.

Figure 1. Expression of 13 Human Proteins Using the ActivePro Kit. DNA templates (12 g) for 13 human proteins were in vitro transcribed/translated in duplicate using the ActivePro Kit and 20 Ci 35S-labeled methionine. A 5 l sample from each reaction was used to calculate yields by TCA precipitation.


Large Scale Reactions with Human Templates
To demonstrate ease of scalability using the ActivePro Kit, reaction volumes ranging from 50 l to 2 ml were assembled and incubated for 1 hr at 37C. Two proteins, caspase 3 and MAPKinase 9, were synthesized in these scaled-up reactions and yields were calculated (Figure 2).

Figure 2. ActivePro Scale-Up Reactions. Caspase 3 and MAPKinase9 DNA templates were transcribed/translated using ActivePro in a range of reaction sizesfrom 50 l to 2 ml. (A) Quantitated protein yields as determined by TCA precipitation. (B) Autoradiograph of the 35S-labeled proteins.


In one hour, several hundred micrograms of protein were obtained in the 1 ml reaction. Nearly 1 mg of human protein was obtained for MAP kinase in the 2 ml reaction. The sharp bands seen in Figure 2 illustrate how the quality of full length protein is not compromised in these scaled-up protocols.


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