UBC Theses and Dissertations
Characterization of human melanotransferrin expressed in recombinant baculovirus infected insect cells Shimizu, Katherine Yumiko
When producing recombinant mammalian proteins in an expression system, the successful completion of posttranslational modifications is an area of concern. One such modification is the attachment of a protein to a glycosyl-phosphatidylinositol (GPI)-anchor in the membrane. In order to investigate this, the baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/'Spodoptera frugiperda Sf9 cells) was used to express the human melanoma-associated antigen, p97 or melanotransferrin. An unusual feature of this protein is its attachment to the cell surface by a GPI-anchor. The expression of p97 at the surface of recombinant virus infected Sf9 cells was shown by FACS analysis using monoclonal antibodies that recognize two different epitopes. Immunoprecipitation of p97 from [35S]-methionine metabolically labelled p97 B-2-I infected Sf9 cells revealed that recombinant p97 is slightly smaller than p97 expressed by the SK-MEL-28 human melanoma cell line and that a soluble form of p97 was present in the spent medium of the infected Sf9 cells. While the GPI-anchored form of recombinant p97 partitioned into the detergent phase upon Triton X-114 extraction, the form found in the spent medium partitioned into the aqueous phase, suggesting that it may be analogous to the secreted form of p97 produced by SK-MEL-28 cells. The glycosylation of recombinant p97 from virus infected Sf9 cells was also analyzed. Although an Endoglycosidase H-resistant form of p97 was detected, it is likely that the processing of N-linked oligosaccharides to the complex-type was incomplete. Finally, the GPI-llinkage of p97 expressed in Sf9 cells was demonstrated by phosphatidylinositol-specific phospholipase C sensitivity and Triton X- 114 extraction. The ability to express GPI-linked proteins in this system will be useful for the bioengineering and commercial production of proteins.