Expression cloning

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The expression cloning includes methods for identification of unknown genes using a property or function of them expressed proteins . By means of cloning , a gene library is first generated in vectors that allow the expression of the DNA. Each clone codes for a maximum of one protein. This expression library is searched for a characteristic of a particular protein or group of proteins. This filtering out or searching is known as screening . The clones isolated in this way are then further characterized.

principle

In most cases, cDNAs are examined and isolated based on the properties of the protein they encode. For this purpose, cDNAs are generated from the mRNA of the organism, the tissue, the cell type or the stage of development, which make the isolation of the desired cDNA probable, and ligated into the cloning site of an expression vector. So was z. B. isolated the first cDNA of a voltage-gated potassium channel from the electrical organ of the marble electric ray . Many of the cDNAs known today have been characterized using the bacteriophage lambda gt11 expression system. The bacteriophage Lambda allows a very dense plating on an E. coli bacterial lawn. Each plaque represents a clone after induction a β-galactosidase - fusion protein generated. The proteins can be detected after the phage has been transferred to a membrane filter (often nitrocellulose ), e.g. B. by means of an antibody against the protein to be examined or, in the case of transcription factors , with a radioactively labeled double-stranded DNA.

Another method uses so-called pools of cDNA clones in vectors that allow in vitro transcription with RNA polymerases from bacteriophages, such as T7 RNA polymerase . After injection, the RNAs are translated into oocytes of the clawed frog Xenopus laevis . The oocytes are checked for the presence of the protein sought, e.g. B. an ion channel or a G protein-coupled receptor is examined. The positive pools are separated into subpools until exactly one RNA produces the desired effect. An example of this strategy is the cloning of the ATP receptor. The yeast two-hybrid system , the yeast one-hybrid system and the phage display are also used for expression cloning.

Individual evidence

  1. ↑ TJ Jentsch, K. Steinmeyer, G. Schwarz: Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. In: Nature. Volume 348, Number 6301, December 1990, pp. 510-514, doi : 10.1038 / 348510a0 , PMID 2174129 .
  2. ^ RA Young, RW Davis: Efficient isolation of genes by using antibody probes. In: Proceedings of the National Academy of Sciences . Volume 80, Number 5, March 1983, pp. 1194-1198, PMID 6219389 , PMC 393560 (free full text).
  3. ^ Y. Ramanathan: Functional Cloning, Sorting, and Expression Profiling of Nucleic Acid-Binding Proteins. In: Genome Research. 12, p. 1175, doi : 10.1101 / gr.156002 .
  4. ↑ DA Melton, PA Krieg, MR Rebagliati, T. Maniatis, K. Zinn, MR Green: Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. In: Nucleic acids research. Volume 12, Number 18, September 1984, pp. 7035-7056, PMID 6091052 , PMC 320141 (free full text).
  5. ↑ CZ Plautz, HC Williams, RM Grainger: Functional Cloning Using a Xenopus Oocyte Expression System. In: Journal of visualized experiments: JoVE. Number 107, January 2016, p. E53518, doi : 10.3791 / 53518 , PMID 26862700 .
  6. ↑ KD Lustig, AK Shiau, AJ Brake, D. Julius: Expression cloning of an ATP receptor from mouse neuroblastoma cells. In: Proceedings of the National Academy of Sciences . Volume 90, Number 11, June 1993, pp. 5113-5117, PMID 7685114 , PMC 46665 (free full text).
  7. ↑ JL Jestin: Functional cloning by phage display. In: Biochemistry. Volume 90, Number 9, September 2008, pp. 1273-1278, doi : 10.1016 / j.biochi.2008.04.003 , PMID 18466773 (review).