Ambion's ActivePro In Vitro Translation Kit, derived from an E.coli lysate, synthesizes higher amounts of protein and proteins with greater biological activity than reticulocyte lysate-based kits and also other commercial E.coli based kits. This results in better performance in biological and functional assays.
Fully Compatible with Eukaryotic Protein Translation
Reticulocyte (retic) lysates have traditionally been the in vitro translation system of choice, especially for researchers working with eukaryotic proteins. However, E.coli derived lysate systems are capable of producing most eukaryotic as well as prokaryotic proteins, often generating considerably higher yields. In prokaryotic transcription, the nascent 5' end of the mRNA becomes available for ribosomal binding, allowing transcription and translation to proceed simultaneously. The early binding of ribosomes to the mRNA improves transcript stability and promotes efficient translation. In Ambion's new ActivePro In Vitro Translation Kit, transcription and translation are likewise coupled and require only a DNA template (either prokaryotic or eukaryotic) with the proper promoter and ribosomal binding sequences. This kit has been engineered to produce both high yields of protein and high levels of functionally active protein from both eukaryotic and prokaryotic derived sequences.
Better Yields and Faster Translation than Reticulocyte Lysate Systems
ActivePro synthesizes protein at a very high rate (several hundred microgram per hour per ml of reaction) with the reaction 80% complete in just 1 hour. In contrast, typical coupled retic kits require 1.5 hours longer incubation. Other major disadvantages of reticulocyte based cell-free systems are their inability to provide preparative amounts of synthesized protein, large amounts of lysates being more expensive and also the need for acute manipulation of animals.
ActivePro's E.coli based highly active lysate, an efficient energy regeneration system, and a large supply of amino acids support faster protein synthesis and higher yields. As seen in Figure 1, translation of a sequence with ActivePro typically yields greater than 1000 fold more protein than with a commercially available coupled retic lysate kit. ActivePro also produces proteins that have significantly higher biological activity than a coupled retic system. Figure 2 de monstrates the higher biological activity of both luciferase and -galactosidase when expressed with ActivePro as compared to a coupled retic system. Similar results were obtained with other proteins tested (data not shown).
Figure 1. The ActivePro Kit vs. Coupled Reticulocyte Lysate Kit . DNA templates (1-2 g) encoding several prokaryotic and eukaryotic (*) proteins were in vitro transcribed/translated using 20 Ci 35S-labeled methionine for 1 hour at 37C according to the ActivePro protocol and for 1.5 hour with the retic. 35S- methionine was measured by TCA precipitation and the total protein yields were calculated.
Figure 2. Biological Activities of Proteins Produced Using ActivePro vs. a Coupled Retic Kit. DNA template (1 g) for -galactosidase (A) or luciferase (B) was used in either an ActivePro Kit reaction or a commercially available coupled retic kit according to protocol. Each reaction included 20 Ci 35S-methionine. Each reaction (1 l) was assayed for -gal by colorimetric ONGP hydrolysis or for luciferase by luminescence.
Higher Biological Activity than Other E.Coli Lysate Systems
Often a translation system produces high levels of protein with only a fraction being functionally active. Since many applications require functional protein, biological activity is often the most important characteristic of the synthesized product. The ActivePro lysate and Activity Enhancer reagent (included in the kit) were specifically developed to increase protein stability and enhance proper folding, resulting in more active prot ein per reaction. Rhodanese (eukaryotic) or -galactosidase (prokaryotic) proteins were synthesized using ActivePro and two other commercial E.coli lysate systems. The reactions were then used in a functional assay specific for each protein to measure total protein activity (units of activity/ml reaction). Figure 3 demonstrates that ActivePro generates proteins with higher activity than other kits.
Figure 3. Biological Activity of Proteins Produced Using the ActivePro Kit vs. Other Commercial Kits. DNA template (1 g) for -galactosidase (-gal) (A) or rhodanese (B) was used in either an ActivePro Kit reaction or other commercially available E.coli lysate kits according to protocol. The reaction (1 l) was assayed for -gal by colorimetric ONGP hydrolysis assay and rhodanese by the colorimetric assay as described by Sorbo1.
Proteins synthesized using ActivePro also show higher specific activity (expressed as activity units/mg protein) when compared to proteins translated in other systems. Figure 4 shows that the specific activity of proteins made with ActivePro was much higher than Kit 1 (a commercially available kit) and may even approach or equal the activity of the native protein (Figure 3, Panel A). Figure 5 demonstrates the sharp bands produced by ActivePro translation, suggesting that full-length translation products are produced. An increase in specific activity indicates an increase in full length active protein. Proteins with high specific activity increase the availability of functional protein for downstream assays. Even though other kits produced high protein yields, these proteins did not show high specific acti vity (data not shown), suggesting that the proteins synthesized were either not full length or not fully functional.
Figure 4. Specific Activity of Proteins Synthesized Using the ActivePro Kit. DNA template (1 g) for -galactosidase (-gal) (A) or rhodanese (B) was used in either an ActivePro Kit reaction or another commercially available E.coli lysate kit according to protocol. Each reaction included 20 Ci 35S-labeled methionine. Each reaction (1 l) along with native -gal (Sigma) was assayed for by colorimetric ONGP hydrolysis assay. Rhodanese was assayed by the colorimetric assay described by Sorbo1. Yield was assayed by TCA precipitation and specific activity was calculated. The specific activity of rhodanese is as reported by Kudlicki2.
Figure 5 . Yields of Both Prokaryotic and Eukaryotic Proteins. DNA templates (1-2 ug) to several prokaryotic and eukaryotic proteins were in vitro transcribed/translated using 20 Ci 35 S-labeled methionine for 1 hour at 37C according to the ActivePro protocol. 5 l of each reaction was assessed on an SDS-PAGE gel and exposed overnight to film.
Efficient Synthesis of Many Prokaryotic and Eukaryotic Proteins
During development of an in vitro translation system, the lysate is optimized to translate the protein(s) used for testing; this is often the "control" provided in a commercially available kit. However, excellent translation of the control does not necessarily mean that the translation system will give the same yields for ot her unrelated proteins. Figure 5 demonstrates that a range of both prokaryotic (GroES, CAT, methionyl-tRNA transformylase (MTF), alkaline phosphatase (AP), SrmB, DEAD, and -gal) and eukaryotic (Green Fluorescent Protein (GFP) and Rhodanese (Rho)) proteins ranging in size from 10-120 kDa can be successfully synthesized using the ActivePro In Vitro Translation Kit.
Easy to Use
ActivePro is easy to use. You simply provide a labeled amino acid (optional) and a DNA template that contains your gene of interest, a T7 promoter, ribosome binding site, and T7 terminator. The DNA template can be easily constructed by inserting your gene of interest into the multiple cloning site of Ambion's control plasmid included in the ActivePro Kit. Simply premix the reagents included in the kit and add your DNA template (plasmid or PCR product) and a labeled amino acid if desired, and incubate at 37C for 1 hour. The resulting biologically active protein is then ready for further functional analysis.
Cat# Product Name Size 1295 ActivePro In Vitro Translation Kit 25 rxns