Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells.
OBJECTIVE
Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein.


RESULTS
HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media.


CONCLUSIONS
RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.
Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Zand Street, Shiraz, Iran.
Hajar
H
Jaberie
Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Zand Street, Shiraz, Iran. fakhraddin.naghibalhossaini@mail.mcgill.ca.|Autoimmune Research Center and Recombinant Protein Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran. fakhraddin.naghibalhossaini@mail.mcgill.ca.
Fakhraddin
F
Naghibalhossaini
D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000737:chemistry; D006031:Glycosylation; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism*
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; C043392:SERPINA1 protein, human; D000515:alpha 1-Antitrypsin
HepG2; Recombinant expression; Transfection; α1-Antitrypsin