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1 Acta Biochimica Polonica

1 2001

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The cell-free protein biosynthesis - applications and analysis of the system

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Lamla T. , Mammeri K. , Erdmann V. A.

Acta Biochimica Polonica

2001

tom 48

nr 2

The in vitro protein biosynthesis has the potentials to be come a powerful technology for biochemical research. Beside the determination of structure and function the in vitro evolution of proteins is also of great interest. The system described was used to produce bo vine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and with out fusion of the Strep-tag II af fin ity pep tide. The proteins were purified after and during protein biosynthesis by using a StrepTactin Sepharose matrix. No significant influence of the Strep-tag and the conditions during the af fin ity chro ma tog ra phy on mat lira tion or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display. The selection of a specific mRNA coding for a short ened FABP with a N-terminal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. Af ter nine cy cles of tran scrip tion, trans la tion, affinity selection and RT-PCR the protein with the His-tag could be enriched 108-fold. In or der to cor re late a pos si ble re la tion ship be tween changes in pro tein pop u la tion and bi o log i cal func tion stud ies were ini ti ated in which 2-dimensional pro tein pat terns of the to tal in vitro sys tem were com pared af ter 0 and 2 h re ac tion time. The very in ter- esting findings are that a number of proteins disappear, while others are newly formed dur ing protein synthesis.