Reconstitution

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Under reconstitution ( . English reconstitution ; lat. 'Re back '(meaning "repetition of an action") and constituo , stand up, build, organize', ie a "re-assembling") is understood in the cell biological and virological aligned Biochemistry the Restoration of original biological activity by bringing together several separate biological (cell) components. First, the biologically active material is fractionated , the presumably required biomolecules and / or molecular complexes (homo- or hetero- oligomers ) are partially or completely purified - whereby the biological activity (functionality) is temporarily lost due to the separation - in order to then convert them to bring together (reconstitute) a simplified, but the natural starting conditions adapted artificial test system . During this process, the original biological activity is restored, can be measured and compared with that of the native system in terms of specificity.

Reconstitution of a native system

Reconstitution goes far beyond a simple enzyme test in its complexity . While a native or - after denaturation - renatured enzyme is examined for its catalytic properties in such a test , a reconstitution allows e.g. For example, the physiological analysis of a plurality of different proteins assembled enzyme complexes , the study of kinetics , substrate specificity, and transport stoichiometry of transmembrane transport systems (with inner and outer ), or the re-assembly of functional virus particles from the individual components.

The following schematic steps are typically followed to achieve native system reconstitution. Biological activity is only present in very specific steps and can then be determined; No activity can be measured in the steps in between:

  1. Selection of the biological material in which the biochemical activity to be studied is found or suspected;
  2. Strict control of the selected biological material for its condition prior to processing;
  3. Digestion of the biological material - possibly while maintaining the intactness of cells or organelles ;
  4. Establishing and performing a suitable test to determine the biological activity to be examined;
  5. Further separation of the biological material e.g. B. by fractional centrifugation and determination of the activity in the supernatants obtained;
  6. Further purification of individual components from the active fractions by chromatographic separation or sedimentation methods ;
  7. Control of the purification through biochemical analysis;
  8. Possibly necessary renaturation of the purified biomolecules;
  9. Any necessary preparation of auxiliary systems (e.g. liposomes );
  10. Gradual or global reconstitution of the natural biological components taking into account the physiological parameters;
  11. Measurement of biological activity in the reconstituted system.

Examples

In the following systems (in a thematically limited selection), a reconstitution of functional biological systems from biomolecules and biomolecular complexes - with or without membrane vesicles (liposomes) - was achieved.

References and comments

  1. ↑ Study of the literature, decision based on the criteria (a) availability, (b) required quantity, (c) frequency of experiments, (d) freshness and stability of the starting material, etc.
  2. ↑ In animals e.g. B. a particular organ ; in plants, a basic organ or organs derived therefrom; for microorganisms or viruses defined fermentations or cell cultures etc.
  3. ↑ Species , subspecies , age, stage of development / growth stage, nutritional criteria , etc.
  4. ↑ Mechanically by repeated freezing and thawing, hypotonic shock, decompression by sonification, Manton-Gaulin homogenizer or French press etc., by shearing (Potter Elvehjem homogenizer, Ultra Turrax, ball mill, etc.) or enzymatically ( pectin , collagenase , zymolyase, lysozyme or similar enzymes) etc.
  5. ↑ Determination of specific activity, d. H. Unit of activity per mg protein (or mg DNA, RNA etc.)
  6. ↑ Native: without or with weak detergents or stabilizers ( protease inhibitors ); denaturing: in the presence of strong detergents and / or chaotropic compounds etc.
  7. ↑ SDS-PAGE , isoelectric focusing etc.
  8. ↑ Physiological buffers , temperature, tonicity, etc.
  9. ↑ B. Hoffmann, A. Stöckl, M. Schlame, K. Beyer, M. Klingenberg: The reconstituted ADP / ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine ​​mutants . (PDF; 591 kB) In: J. Biol. Chem. , Vol. 269 (3), 1994, pp. 1940-1944
  10. ↑ XS Xie: Reconstitution of ATPase activity from individual subunits of the clathrin-coated vesicle proton pump. The requirement and effect of three small subunits . In: J. Biol. Chem. , 271 (48), 1996, pp. 30980-30985, PMID 8940086
  11. ↑ P. Mueller, DO Rudin, H. Ti Tien, WC Wescoytt: Reconstitution of excitable cell membrane structure in vitro . In: Circulation , No. 26, 1962, pp. 1167-1171
  12. ^ H. Fraenkel-Conrat, B. Singer: Virus reconstitution and the proof of the existence of genomic RNA . In: Phil. Trans. R. Soc. Lond. , B 354, pp. 583-586, PMID 10212937
  13. ↑ KA Hutchison, BK Brott, JH De Leon, GH Perdew, R. Jove, WB Pratt: Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system . In: J. Biol. Chem. , 267 (5), 1992, pp. 2902-2908, PMID 1310678