Shuffling of heterologous dna sequences

C - Chemistry – Metallurgy – 12 – N

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C12N 15/10 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2284097

The present invention relates to a new method of shuffling especially heterologous polynucleotide sequences, screening and/or selection of new recombinant proteins resulting therefrom having a desired biological activity, and especially to production and identification of novel proteases exhibiting desired properties. The method comprises the following steps; i) identification of at least one conserved region between the heterologous sequences of interest; ii) generating fragments of each of the heterologous sequences of interest, wherein said fragments comprise the conserved region(s), in a preferred embodiment due to the use of parts of the regions(s) as primers; and iii) shuffling/recombining said fragments using the conserved region(s) as (a) homologous linking point(s).

Cette invention, qui a trait à une nouvelle méthode de réarrangement de séquences de polynucléotides hétérologues, de criblage et/ou de sélection de nouvelles protéines de recombinaison qui en sont issues, dotées d'une action biologique souhaitée, porte également sur la production et l'identification de nouvelles protéases présentant des propriétés désirées. La méthode comprend les étapes suivantes: (i), identification d'au moins une région conservée entre les séquences hétérologues concernées, (ii), production de fragments de chacune de ces séquences, lesdits fragments comportant la ou les régions conservées - dans une réalisation préférées du fait de l'utilisation de parties de la région ou des régions en tant qu'amorce(s) - et (iii), réarrangement/recombinaison desdits fragments au moyen de la région ou des régions conservée(s) en tant que point(s) homologue(s) de liaison.

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