INFLUENCE OF DIFFERENT SIGNAL PEPTIDES AND PROSEQUENCES ON EXPRESSIONAND SECRETION OF HUMAN TISSUE-PLASMINOGEN ACTIVATOR IN THE BACULOVIRUS SYSTEM

Foreign secretory pathway proteins are often produced in surprisingly low amounts in the baculovirus/insect cell expression system. One poss ible reason for this is that heterologous signal peptides might be ine fficiently recognized by the insect cell protein translocation machine ry. This idea was supported by a recent study showing that secretion o f a plant protein in the baculovirus system was enhanced when its sign al peptide was replaced with an insect-derived signal peptide (Tessier , D. C., Thomas, D. Y., Khouri, H. E., Laliberte, F., and Vernet, T. ( 1991) Gene (Amst.) 98, 177-183). We have extended these observations b y measuring the effects of different signal peptide and signal peptide -prosequence combinations on baculovirus-mediated expression and secre tion of human tissue plasminogen activator (t-PA). Replacement of the native prepropeptide with signal peptides from a lepidopteran insect s ecretory protein (cecropin B), a major baculovirus structural glycopro tein (64K), or an abundant, highly conserved lumenal protein of the ro ugh endoplasmic reticulum (GRP78/BiP, a 78-kDa glucose-regulated prote in/immunoglobulin heavy chain-binding protein), had no significant eff ect on t-PA expression or secretion. The same results were obtained wi th the signal peptide from honeybee prepromellitin, which was able to enhance secretion of plant propapain (Tessier et al., 1991 (above)). S imilar results were obtained when heterologous signal peptides were co mbined with the native prosequence or when the intact cecropin B prepr osequence was used. Translational initiation at an upstream, in-frame ATT, which could functionally inactivate any signal peptide, did not e xplain the low efficiency of t-PA secretion. Finally, deletion of the native signal peptide, prosequence, or both, failed to increase t-PA p roduction. These results showed that insect-derived signal peptides an d/or prosequences cannot always enhance the expression and/or secretio n of foreign secretory pathway proteins in the baculovirus system. The y also suggested that the inability of insect cells to recognize the p rocessing signals in human t-PA efficiently is probably not the major factor preventing its high level production in this system.