Expression of murine soluble CD4 protein in baculovirus infected insect cells.
The expression of murine soluble CD4 (L3T4) protein (sCD4) by baculovirus-infected insect cells was characterized. The yield of sCD4 reached 2 mg/l culture supernatant late in infection. Nevertheless, a large amount of sCD4 remained cell-associated, presumably in the endoplasmic reticulum or an early golgi compartment, as indicated by the endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitivity of its carbohydrate chains. The secreted form of sCD4 is modified with both endo-beta-N-acetyl-D-glucosaminidase D (endo-D) and endo-H-sensitive oligosaccharides. It was possible that the incomplete secretion indicated faulty glycosylation or improper folding of the sCD4 protein. However, inhibitor studies showed that complete carbohydrate processing is not required for secretion of sCD4 by insect cells. Moreover, maintained reactivity with a panel of monoclonal Ab as well as phase partitioning experiments suggested that secretion is apparently not caused by misfolding of the sCD4 protein. Similar results were obtained with biologically active murine interleukin-4 produced by insect cells. This indicates that an inefficient secretory pathway may be a general problem of baculovirus-infected insect cells and is not a consequence of incorrect molecular conformation.
Kupsch
J
J
Max-Planck-Institut für Immunbiologie, Freiburg, Germany.
Saizawa
K M
KM
Eichmann
K
K
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015704:CD4 Antigens / Q000096:biosynthesis* / Q000737:chemistry*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D007313:Insecta / Q000235:genetics / Q000378:metabolism*; D015847:Interleukin-4 / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis; D017026:Swainsonine / Q000494:pharmacology; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D015704:CD4 Antigens; D011994:Recombinant Proteins; D014415:Tunicamycin; D015847:Interleukin-4; D017026:Swainsonine