Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.
The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH.
Zajc
Tajana
T
Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Suban
Dejan
D
Rajković
Jelena
J
Dolenc
Iztok
I
D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D046508:Culture Techniques; D004151:Dipeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000821:virology
D004151:Dipeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C026134:glutamate carboxypeptidase