Cloning, sequencing, expression, and purification of the C isozyme of mouse phosphofructokinase.
The cDNA of mouse phosphofructo-1-kinase isozyme C was cloned and sequenced. The coding region translates into a protein of 85,473 Da containing 785 amino acids. The cDNA includes 57 base pairs of a 5'-untranslated region and a 3' untranslated region of 284 base pairs containing a polyadenylation signal, AUUAAA, located 17 bases upstream from the poly(A) tail. The cDNA was ligated into a pET vector and transformed into a pfk(-) strain of Escherichia coli (DF1020) that contained the pLysS plasmid and an integrated lambda DE3 prophage that includes a single copy of the gene for T7 RNA polymerase under control of the inducible LacUV5 promoter. Conditions for maximum induction of soluble enzyme activity was developed to produce up to 2400 units of soluble enzyme activity per liter of growth medium. The enzyme could be purified to homogeneity with a yield of approximately 60% by a single purification step on ATP-Sepharose.
Gunasekera
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Department of Biochemistry and Molecular Biology, Chicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064, USA.
Kemp
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D007527:Isoenzymes; D011994:Recombinant Proteins; D010732:Phosphofructokinase-1