Construction of a strain-producer of the chimeric protein consisting of RNA polymerase and a DNA-affinity domain

Abstract

One of the recent perspective trends of molecular biotechnology is cell-free synthesis of protein. The procedure of cell-free synthesis of protein is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes. Procreation of the transcription stage requires participation of specific RNA polymerase which initiates process of mRNA synthesis from the particular sites of recognition. Often the DNA-dependent RNA polymerase of a bacteriophage of T7 (T7 RNA polymerase) is for this purpose applied. For improvement of qualitative characteristics of the T7 RNA polymerase in the real work the new strain of Escherichia coli producing this enzyme fused with the DNA-affine Sso7d domain of a thermophilic bacterium Sulfolobus solfataricus is created. The producing ability of the received recombinant strain concerning synthesized chimera protein reaches 625 un/l of cultural liquid, and the specific activity of the purified enzyme preparation was 80 un/ μg of protein. The received enzyme is intended for use as tools at synthesis of proteins in cell-free system.