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 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.
Marcillat
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UFR Chimie-Biochimie, Université Claude Bernard Lyon I, UPRESA CNRS 5013, 43 Boulevard du 11 Novembre 1918, Villeurbanne, F-69622, France.
Perraut
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C
Granjon
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Vial
C
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Vacheron
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics* / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D003402:Creatine Kinase