Electrophoretic and isoelectric focusing analysis of human recombinant alpha 2-HS glycoprotein produced in insect cells: analysis of the post-translational events.
Alpha 2-HS glycoprotein (AHSG) is a human serum glycoprotein synthesized by liver cells. It is a natural inhibitor of the insulin receptor tyrosine kinase activity. We produced this protein in insect cells by using a recombinant baculovirus expressing the whole coding sequence of the protein. By analyzing AHSG on isoelectric focusing and on sodium dodecyl sulfate (SDS) gels, followed by immunoblot, AHSG produced in insect cells was found to be phosphorylated and to possess the connecting peptide between the A and the B chains. The same features were found in the protein produced by Hep3B, a human liver cell line that synthesizes AHSG. By contrast, no phosphorylation could be detected in AHSG present in normal human plasma, and the connecting peptide was clipped. As the protein produced in insect cells is active on insulin receptors, in contrast to the plasma protein, our results suggest that the biological activity of the protein may be associated with its single chain form together with its phosphorylation.
Kalabay
L
L
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston 29464, USA.
Mathur
S
S
Bobin
S
S
Arnaud
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000032:analysis* / Q000235:genetics; D002460:Cell Line; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D018411:Spodoptera; D014407:Tumor Cells, Cultured; D060749:alpha-2-HS-Glycoprotein
C556354:AHSG protein, human; D001798:Blood Proteins; D011993:Recombinant Fusion Proteins; D060749:alpha-2-HS-Glycoprotein