Cell-free gene expression

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The cell-free expression ( in vitro - gene expression , cell-free protein synthesis, in vitro - translation ) is an applied laboratory method for the production of proteins , most of the recombinant proteins.

properties

The integrity of a living cell is not required for the biosynthesis of proteins . The transcription and translation apparatus of a lysed cell can also be used. Energy carriers and amino acids are added via a membrane system.

The greatest advantage of this method over synthesis in cells is that toxic proteins can also be synthesized and non-proteinogenic amino acids can be introduced. Compared to chemical protein synthesis, it is characterized by the simpler production of long amino acid chains. Both methods, cell-free gene expression and chemical synthesis, have their advantages and disadvantages. B. length, amino acid sequence and quantity of protein obtained.

On a laboratory scale, the proteins are often produced in lysates or extracts from E. coli , insect cells , wheat germ or mammalian cell extracts from K562 cells , CHO cells or reticulocytes from rabbits. The extracts are incubated with an RNA, usually generated by means of in vitro transcription , and an amino acid mixture in a suitable buffer system . The proteins produced can then be detected in the Western Blot and, depending on the protein, their properties, such as the substrate turnover in enzymes or the DNA interaction in transcription factors , can be examined. In order to detect the synthesized proteins, an amino acid can also be replaced by a radioactively labeled (mostly 35 S-methionine) or an amino acid labeled with a fluorescent dye . The corresponding translation extracts are available as kits from various suppliers. A disadvantage of the method is the risk of RNA degradation by RNases . For this reason, and because of the associated time savings, systems in which transcription and translation are coupled have become established. In addition to amino acids and buffers, the commercially available extracts are mixed with the appropriate RNA polymerase from bacteriophages (T3, T7 , SP6) and the desired DNA template, a plasmid vector that contains at least one suitable promoter flanking the multiple cloning site .

In Germany a. a. the Fraunhofer-Gesellschaft is working on the further development of cell-free gene expression as part of the BMBF strategy process "Biotechnology 2020+". The aim is to develop a modular bioreactor for efficient cell-free protein synthesis.

Alternatively, a protein can partially be generated in vitro by chemical Merrifield synthesis of peptides and subsequent protein ligation .

literature

  • Lutz Essers: Renaissance of cell-free protein biosynthesis. In: GENOMXPRESS 4.08 (PDF; 8.2 MB), pp. 13–15 (review article)

Individual evidence

  1. ^ T. Terada, T. Murata, M. Shirouzu, S. Yokoyama: Cell-free expression of protein complexes for structural biology. In: Methods in molecular biology. Volume 1091, 2014, pp. 151-159, doi : 10.1007 / 978-1-62703-691-7_10 , PMID 24203330 .
  2. ^ AK Brödel, DA Wüstenhagen, S. Kubick: Cell-free protein synthesis systems derived from cultured mammalian cells. In: Methods in molecular biology. Volume 1261, 2015, pp. 129-140, doi : 10.1007 / 978-1-4939-2230-7_7 , PMID 25502197 .
  3. ↑ Dmitriy A. Vinarov, Carrie L. Loushin Newman, Ejan M. Tyler, John L. Markley, Mark N. Shahan: Wheat Germ Cell-Free Expression System for Protein Production. In: Current Protocols in Protein Science (2006). Volume 44, Issue 1, pp. 5.18.1-5.18.18. doi: 10.1002 / 0471140864.ps0518s44 .
  4. ↑ Fraunhofer Institute : System research project cell-free bioproduction .