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Translate High Yields of Biologically Active Proteins

ActivePro In Vitro Translation Kit


Cell-free translation systems are a convenient method for synthesizing proteins in vitro. The major advantages of these in vitro translation systems over conventional in vivo systems are greater flexibility, faster reaction times, and less labor-intensive protocols.

Ambions ActivePro In Vitro Translation Kit is a cell-free, E. coli based translation kit that excels at expressing a wide range of functional proteins for a variety of downstream applications. ActivePro generates high yields of proteins with higher biological activity compared to other commercially available kits. This report demonstrates that a number of complex human proteins can be efficiently translated using this technology.


Performance with Human Templates
Thirteen human full length, open reading frame (ORF) clones were selected from the NIH Mammalian Gene Collection (MGC). The corresponding full length sequences were subcloned into Ambions optimized translation vector (included in the ActivePro Kit) and sequences were confirmed. 12 g each of the 13 constructs were translated in a standard 50 l ActivePro reaction. Each reaction included 35S-labeled methionine and was incubated for 1 hour at 37C. Protein yields, as determined by TCA precipitation, were calculated based on the number of labeled methionines in each sequence. 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.


Protein Expression With ActivePro Surpasses Competitors
To compare the efficiency of ActivePro with two competing systems, five human templates were expressed using the manufacturers protocols. Both yields (Figure 3) and biological activities of the reaction products (Figure 4) were assayed. ActivePro gave the highest yields for all five of the proteins translated. And in all but one case (DHFR), ActivePro-translated proteins showed the highest enzymatic activity.

Figure 3. Comparison of Protein Yields Across E. coli Lysate Kits. DNA templates for 5 human proteins were in vitro transcribed/translated using Ambions ActivePro Kit and two other commercially available kits, according to protocol. The proteins analyzed were dihydrofolate reductase (DHFR), adenylate kinase 1 (AK1), carbonic anhydrase 2 (CA2), caspase 3 (CASP3), and casein kinase 1 (CK1). (A) Quantitated protein yields as determined by TCA precipitation. (B) Autoradiographs of the 35S-labeled proteins.

Figure 4. Comparison of Biological Activity Across E. coli Lysate Kits. The proteins as produced from Figure 3 were tested in functional assays. Relative activities from protein preparations synthesized using two commercially available kits were compared to that from the ActivePro Kit. Dihydrofolate reductase (DHFR): Activity was measured with a standard DHFR assay, which follows the rate of conversion of dihydrofolate and NADPH to tetrahydrofolate and NADP+ through the successive decrease of absorbance of NADPH at 340 nm at 30C [1]. Adenylate kinase 1 (AK1): Activity was measured by a luciferase-based ATP bioluminescence assay (Sigma) in the presence of 1 mM ADP [2]. Carbonic anhydrase 2 (CA2): Esterase activity of CA2 was monitored by the net increase in absorbance at 348 nm in a p-nitrophenyl acetate hydrolysis assay [3]. Caspase 3 (CASP3): Catalytic activity was determined by measuring the proteolytic cleavage of the fluorogenic substrate Ac-DEVD-AFC for 1 hr in a 96 well plate reader (400 nm excitation wavelength, 505 nm emission wavelength). Casein kinase 1 (CK1): Activity was determined by phosphorylation of casein kinase 1 substrate peptide (Upstate).


Summary
ActivePro is a superior, cell-free in vitro translation kit delivering both high yields and active protein. It is fast, easy to use, and scalableas much as 1 mg of protein can be synthesized in just 1 hour. Compared to similar kits, ActivePro consistently expresses more biologically active protein in extremely high yields. Because it does not require special equipment, it is the fastest and most economical kit for producing milligram quantities of protein suitable for any downstream application.


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Ordering Information
Cat# Product Name Size 1295 ActivePro In Vitro Translation Kit 25 rxns
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