The feasibility of the preparation of a purified human enzyme as a reference material standard
Recently developed methodologies offer two possibilities for the large scale production of human proteins and enzymes : - the cloning of the cDNA sequences of the protein of interest and their expression in an heterologous system (in prokaryotic or eukaryotic cells); - the large scale culture of cells naturally expressing high amounts of the protein or the enzyme in question. The cDNAs of gamma-glutamyltransferase were cloned in frame in bacterial or yeast expression vectors. To obtain the expression of rat kidney or Hep G2 GGT cDNA in E.coli or in yeast, plasmids containing the cDNA sequences coding for various parts of GGT were constructed. Transformation of E.coli cells by these hybrid vectors resulted in the production of unglycosylated recombinant proteins, immunologically recognised by specific antibodies. Plasmids expressing the complete coding sequence of rat renal GGT cDNA allowed the production of enzymatically active proteins localised in the periplasmic space, while the expression of the same cDNA sequence encoding the GGT without its NH(2)-terminal region resulted in the production of cytoplasmic proteins. Also, transfection of S.cerevisiae by a vector containing a cDNA of rat renal GGT resulted in expression of immunologically recognised and enzymatically active protein. The human hepatoma cell line Hep G2 expresses relatively high levels of active GGT. The feasibility of high yield cell production by culturing the Hep G2 cells on Cytodex microcarriers was evaluated. Some of the culture conditions on the Cytodex 3 microcarriers were optimised and high cell levels and GGT activity were obtained with cells cultured in the presence of 5% fetal calf serum in the culture medium. The purification of the enzyme from these cells was performed using an ion exchange chromatography in a HPLC system.
Bibliographic Reference: EUR 12842 EN (1990) 47 pp., MF, ECU 4, blow-up copy ECU 6.25
Record Number: 199011132 / Last updated on: 1994-12-01
Original language: en
Available languages: en