Deletion of the GPI pre-anchor sequence in human p97--a general approach for generating the soluble form of GPI-linked proteins.
Melanotransferrin, also named p97, belongs to the transferrin-like group of iron-binding proteins. Unlike the other members of this family, p97 exists in two forms-one soluble form and one attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The GPI-linked form plays a role in the uptake of iron, while the soluble form of p97 has the unique ability of traversing the blood-brain barrier and may be utilized to deliver drug conjugates into the brain. To investigate these possibilities, a recombinant soluble form of p97 from the GPI-linked p97 protein is required. The approach involved sequential deletions of the p97 GPI pre-anchor sequence (PAS) up to the putative site of cleavage/attachment, releasing p97 from attachment to the GPI-anchor and rendering it soluble. Transfection of the p97 deletion constructs into both the CHO and BHK TK(-) cells was performed with the aim of optimizing the production of p97 by utilizing the cell characteristics unique to each cell line. Altering the GPI PAS resulted in the generation of a recombinant soluble form that was secreted at significantly higher rates than from the full-length expressing cell lines. Increases were from 22 x 10(-9) to 241 x 10(-9)microg/cell/h for expression in the CHO cell system and from 220 x 10(-9) to 4970 x 10(-9)microg/cell/h for the BHK system. Furthermore, there appeared to be differences in the secretion rates between the various deletions suggesting the need for closer examination of the C-terminus in achieving maximum production of the altered proteins. The results of this study are likely applicable for expressing soluble forms of other GPI-linked proteins.
Yang
Joseph
J
The Biotechnology Laboratory, Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Tiong
Jacqueline
J
Kennard
Malcolm
M
Jefferies
Wilfred A
WA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D016466:CHO Cells; D002457:Cell Extracts / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D058950:Melanoma-Specific Antigens; D008565:Membrane Proteins / Q000032:analysis; D008647:Mesocricetus; D008715:Methionine / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport / Q000235:genetics; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection; D019287:Zinc Sulfate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D002457:Cell Extracts; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D058950:Melanoma-Specific Antigens; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D019287:Zinc Sulfate; D008715:Methionine; D006026:Glycoside Hydrolases