Cloning, Escherichia coli expression, and phase-transition chromatography-based purification of recombinant rabbit heart mitochondrial creatine kinase

A cDNA clone of the mitochondrial sarcomeric creatine kinase cDNA was obtai ned by screening a rabbit heart library. This cDNA is characterized by a 12 57-nucleotide open reading frame encoding a 419-amino-acid protein with a c leavable 39-amino-acid mitochondrial presequence (Accession No. AJ011334). This new member of the guanidino kinase family shows a high degree of seque nce similarity with the other phosphagen kinases sequenced so far. The matu re 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. (C ) 1999 Academic Press.