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Abstract
In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.
Highlights
In vitro protein biosynthesis became a powerful technology for biochemical research
The limitations caused by ultrafiltration membranes during product removal from a CFCF reactor should be overcome employing the affinity system described here
We fused the two Strep-tag versions I and II to the C terminus of the CAT gene and Strep-tag II to the FABP gene by PCR methods and cloned them into plasmids containing all elements for an efficient in vitro transcription/ translation. We have chosen these two genes, because FABP is a well known standard in our laboratory, and CAT reveals the influence of the Strep-tag on the activity of the fused protein on the basis of its enzymatic activity [16]. It was not known whether the additional 33 and 30 bp encoding the Strep-tag I and II, respectively, would influence the in vitro expression of the new genes, if the fused peptide would disturb the native structure of the proteins, and if the tag would be accessible for affinity chromatography
Summary
EFFICIENT PRODUCT ISOLATION DURING IN VITRO PROTEIN BIOSYNTHESIS VIA AFFINITY TAG*. In vitro protein biosynthesis became a powerful technology for biochemical research. In most cases the use of a synthesized protein for further applications depends on its purity For this purpose the in vitro production and purification of proteins with short affinity tails was established. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The use of a synthesized protein for further applications like crystallization or NMR studies depends to a great extent upon its purity For this purpose, the recombinant production and purification of proteins with short affinity tails have gained widespread application in biotechnology [5]. It was shown that Strep-tag I allows single-step protein purification from bacterial expression systems [7], but its fusion to recombinant proteins is restricted to the C terminus Another variant, designated Strep-tag II, was introduced [8] that did not show this limitation. The limitations caused by ultrafiltration membranes during product removal from a CFCF reactor should be overcome employing the affinity system described here
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