Circular permutant gfp insertion folding reporters

Details Circular permutant gfp insertion folding reporters Circular permutant gfp insertion folding reporters

C - Chemistry, Metallurgy

12

P

C12P 21/06 (2006.01) C07K 14/705 (2006.01)

Patent

CA 2642911

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

La présente invention concerne des procédés portant sur l'essai et l'amélioration du repliement des protéines au moyen de permutants circulaires de protéines fluorescentes, y-compris des permutants circulaires de variantes de GFP et certains de leurs combinaisons. L'invention concerne également diverses molécules d'acides nucléiques et des vecteurs incluant de telles molécules d'acides nucléiques, comprenant des polynucléotides codant des permutants circulaires de protéines fluorescentes dérivées du super-replieur GFP. En l'occurrence, ces polynucléotides comportent un site interne de clonage dans lequel il est possible d'insérer un polynucléotide hétérologue en chaîne avec la séquence codante du permutant circulaire. Quand ils sont exprimés, ces polynucléotides sont capables de rendre compte de la qualité avec laquelle un polypeptide codé par un tel polynucléotide hétérologue inséré est replié en corrélation avec le degré de fluorescence dont il fait preuve.

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