Navigation Links
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 sequen 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.

Protein Express ion 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 bioluminescen ce 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).

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.

back to top

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


Page: All 1 2 3 4

Related biology technology :

1. Translate High Yields of Active Protein
2. Translate More Protein with Higher Biological Activity
3. Yeast Protein Production System Features High Yields and One-Step Purification
4. ArrayScript RT: Maximize Yields of Full-length cDNA
5. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
6. Synthesize High Yields of Biotinylated aRNA
7. Production of Full-Length, Biologically Active MEK1WT and MEK1CA
8. Highly Active and Removable AffinityTagged Lambda Phosphatase
9. Rapid PCR* Using Active Tube Temperature Control On Thermos PCR Machines
10. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
11. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
Post Your Comments:

(Date:11/25/2015)... 25, 2015 Orexigen® Therapeutics, Inc. (Nasdaq: ... a fireside chat discussion at the Piper Jaffray 27th ... . The discussion is scheduled for Wednesday, December 2, ... .  A replay will be available for 14 days ... , Julie NormartVP, Corporate Communications and Business Development , ...
(Date:11/24/2015)... N.J. (PRWEB) , ... November 24, 2015 , ... The ... the recipient of the 2016 USGA Green Section Award. Presented annually since 1961, the ... through his or her work with turfgrass. , Clarke, of Iselin, N.J., ...
(Date:11/24/2015)... ... November 24, 2015 , ... Copper is an ... is bound to proteins, copper is also toxic to cells. With a $1.3 ... Institute (WPI) will conduct a systematic study of copper in the bacteria Pseudomonas ...
(Date:11/24/2015)... 2015 --> ... "Oligonucleotide Synthesis Market by Product & Services (Primer, Probe, ... DNA, RNAi), End-User (Research, Pharmaceutical & Biotech, Diagnostic Labs) ... market is expected to reach USD 1,918.6 Million by ... CAGR of 10.1% during the forecast period. ...
Breaking Biology Technology:
(Date:10/29/2015)... Va. , Oct. 29, 2015 Daon, ... today that it has released a new version of ... customers in North America have ... IdentityX v4.0 also includes a FIDO UAF certified ... are already preparing to activate FIDO features. These customers ...
(Date:10/29/2015)... October 29, 2015 NXTD ... company focused on the growing mobile commerce market ... that StackCommerce, a leading marketplace to discover and ... Wocket® smart wallet on StackSocial for this holiday ... or the "Company"), a biometric authentication company focused ...
(Date:10/27/2015)... 2015 Munich, Germany ... technology (ASGM) automatically maps data from mobile eye tracking ... , so that they can be quantitatively analyzed with ... Munich, Germany , October 28-29, 2015. SMI,s Automated ... mobile eye tracking videos created with SMI,s Eye ...
Breaking Biology News(10 mins):