Construction of a cDNA clone containing the entire coding region of the human liver-type phosphofructokinase

Abstract

Genomic fragments containing coding sequences of the human liver phosphofructokinase (PFKL) were used to screen cDNA libraries and several clones corresponding to PFKL were isolated. Three overlapping cDNA clones spanning the entire coding region were stitched together yielding the plasmid pG-cPFKL3.0 containing a 3Kb PFKL cDNA down stream of the T7 promoter. To assess its coding capacity pG-cPFKL3.0 was transcribed by T7 RNA polymerase and the RNA product was used to program an in vitro translation system. An 80KDa polypeptide which co-migrated with an in vivo labeled PFKL and was recognized by anti PFKL antibodies was synthesized. These results show that the cDNA clone pG-cPFKL3.0 contains the entire coding region of the human PFKL.