Method of disulfide crosslink forming in vitro protein...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/09 (2006.01) C12P 21/02 (2006.01) C12Q 1/26 (2006.01)

Patent

CA 2564831

A process for producing a protein having disulfide bonds easily with high efficiency by the use of a reconstituted protein synthesizing system. It has been found that in place of the use of a cell extract, or crude fraction thereof, wherein due to the presence of various substrates and enzymes for maintaining of a state of reduction including thioredoxin reductase [EC1.6.4.5] and glutaredoxin reductase [EC1.6.4.2], conditioning of a state of oxidation and reduction is difficult, with the use of a reconstituted protein synthesizing system composed of purified components wherein any substrates and enzymes influencing the state of oxidation and reduction are removed, active protein can be efficiently synthesized by means of a reconstituted protein synthesizing system having the state of oxidation-reduction equilibrium between disulfide and thiol artificially regulated.

Procédé servant à produire facilement et avec un rendement de production élevé une protéine ayant des liaisons disulfure grâce à l'utilisation d'un système synthétisant des protéines reconstitué. On a trouvé qu'à la place de l'utilisation d'un extrait de cellule, ou d'une fraction brute de celui-ci, dans lequel à cause de la présence de différents substrats et enzymes servant à maintenir un état de réduction comprenant de la thiorédoxine réductase [EC1.6.4.5] et de la glutarédoxine réductase [EC1.6.4.2], le conditionnement d'un état d'oxydation et de réduction est difficile, grâce à l'utilisation d'un système synthétisant des protéines reconstitué composé de composants purifiés dans lequel tous les substrats et enzymes quels qu'ils soient ayant une incidence sur l'état d'oxydation et de réduction sont enlevés, on peut synthétiser de façon efficace une protéine active au moyen d'un système synthétisant des protéines reconstitué ayant l'état d'équilibre d'oxydoréduction entre le disulfure et le thiol artificiellement régulé.

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