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Cloning, Escherichia coli expression, and phase-transition chromatography-based purification of recombinant rabbit
O Marcillat1, C Perraut, T Granjon
1UFR Chimie-Biochimie, Université Claude Bernard Lyon I, UPRESA CNRS 5013, 43 Boulevard du 11 Novembre 1918, Villeurbanne, F-69622, France.
Abstract:
A cDNA clone of the mitochondrial sarcomeric creatine kinase cDNA was obtained by screening a rabbit heart library. This cDNA is characterized by a 1257-nucleotide open reading frame encoding a 419-amino-acid protein with a cleavable 39-amino-acid mitochondrial presequence (Accession No. AJ011334). This new member of the guanidino kinase family shows a high degree of sequence similarity with the other phosphagen kinases sequenced so far. The mature enzyme was efficiently expressed in Escherichia coli BL21(DE3) cells as a soluble octameric protein using the pET21 plasmid and purified by a three-step improved method including a final phase-transition chromatography.