Human Cell-Free Protein Expression System and vectors

The Human Cell-Free Protein Expression System from Takara Bio is easy to use. The single-tube reaction is easily assembled and protein synthesis is complete in as little as 1 hr at 32°C. The Human Cell-Free Protein Expression System provides high yield (e.g., 50 μg/ml of human eIF4G) of functional protein, including proteins requiring modifications such as glycosylation, phosphorylation, or fatty acylation and even with large proteins (over 150 kDa). Genes of interest may be cloned rapidly into pT7-IRES vectors using In-Fusion cloning technology. After expression, proteins with N-terminal or C-terminal His tag or N-terminal Myc tag can be generated, depending on choice of vector. 

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Principle of the Human Cell-Free Protein Expression System

Principle of the Human Cell-Free Protein Expression System. A) In an easy and simple protocol, target protein translation is initiated by adding pT7-IRES Vector containing the target gene cassette and other kit components. B) The target gene RNA transcribed from the pT7-IRES Vector has an IRES sequence designed to promote protein translation initiation. As protein synthesis progresses, the translation initiation factor from the cell lysate becomes inactivated. The translation enhancement factor in the reaction mixture, however, reactivates this inactivated translation initiation factor and thereby maintains high translation level.

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Time course of beta-galactosidase expression and effect of translation enhancement factor

Time course of beta-galactosidase expression and effect of translation enhancement factor. Eight replicates of beta-galactosidase in vitro translation were performed using 1 µl of Control Vector per reaction. At each of the indicated time points (0, 0.5, 1, 2, 3, 4.5, 6, and 8 hours after start of the reaction), one reaction tube was removed and used for beta-galactosidase activity assay with O-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. A separate set of reactions were conducted in absence of the translation enhancement factor (Mixture-2). The activity of beta-galactosidase increased over time to peak at approximately 4.5 h. Additionally, the presence of Mixture-2 containing translation enhancement factor markedly increased yield of active protein.

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Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System

Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System. In vitro translation reactions were performed to synthesize human Dicer (200 kDa, lane 2) or human eIF4G (170 kDa, lane 3) protein. Reactions were analyzed by SDS-PAGE and coomassie blue staining. Arrowheads indicate target proteins. Lane 1, negative control.

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3281: Human Cell-Free Protein Expression System

pT7-IRES DNA series are expression vectors designed for use with the Human Cell-Free Protein Expression System. pT7-IRES vectors contain tag sequences such as His-Tag or c-Myc Tag, Factor Xa cleavage site, Multiple Cloning Site (MCS), polyA, and T7 terminator are located at the downstream of a T7 promoter and an EMCV IRES. pT7-IRES His-N DNA is an expression vector including a his-tag sequence. Using pT7-IRES His-N DNA together with the Human Cell-Free Protein Expression System (Cat. #3281) enables the synthesis of your target protein as an N-terminal fusion with a his tag. A Factor Xa cleavage site is included so that the his tag can be removed from the synthesized fusion protein. Any target gene cloned into the MCS in frame is transcribed as an RNA-containing-EMCV IRES under the control of the T7 promoter. Efficient high level protein synthesis can be performed in the Human Cell-Free Protein Expression System, due to the effect of the EMCV IRES, which promotes protein translation initiation.

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3290: pT7-IRES His-N DNA

pT7-IRES DNA series are expression vectors designed for use with the Human Cell-Free Protein Expression System. pT7-IRES vectors contain tag sequences such as His-Tag or c-Myc Tag, Factor Xa cleavage site, Multiple Cloning Site (MCS), polyA, and T7 terminator are located at the downstream of a T7 promoter and an EMCV IRES. pT7-IRES His-C DNA is an expression vector including a his-tag sequence. Using pT7-IRES His-C DNA together with the Human Cell-Free Protein Expression System (Cat. #3281) enables the synthesis of your target protein as a C-terminal fusion with a his tag. A Factor Xa cleavage site is included so that the his tag can be removed from the synthesized fusion protein. Any target gene cloned into the MCS in frame is transcribed as an RNA-containing-EMCV IRES under the control of the T7 promoter. Efficient high level protein synthesis can be performed in the Human Cell-Free Protein Expression System, due to the effect of the EMCV IRES, which promotes protein translation initiation.

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3291: pT7-IRES His-C DNA

pT7-IRES DNA series are expression vectors designed for use with the Human Cell-Free Protein Expression System. pT7-IRES vectors contain tag sequences such as His-Tag or c-Myc Tag, Factor Xa cleavage site, Multiple Cloning Site (MCS), polyA, and T7 terminator are located at the downstream of a T7 promoter and an EMCV IRES. pT7-IRES Myc-N DNA is an expression vector including a c-Myc tag sequence. Using pT7-IRES Myc-N DNA together with the Human Cell-Free Protein Expression System (Cat. #3281) enables the synthesis of your target protein as an N-terminal fusion with a c-Myc tag. A Factor Xa cleavage site is included so that the c-Myc tag can be removed from the synthesized fusion protein. Any target gene cloned into the MCS in frame is transcribed as an RNA-containing-EMCV IRES under the control of the T7 promoter. Efficient high level protein synthesis can be performed in the Human Cell-Free Protein Expression System, due to the effect of the EMCV IRES, which promotes protein translation initiation.

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3292: pT7-IRES Myc-N DNA