Construct for Assessment of Transfected Secretory Proteins Using an Independently Secreted Reporter Gene

Furin is a membrane-associated endopeptidase located in the trans-Golgi network that has the enzymatic capabilities to process a large number of precursors (6,11). Furin catalyzes the cleavage of a specific sequence (R-6X-5-R-4-X-3-R/Lys-2-R-1) with the added condition that there is no hydrophobic aliphatic amino acid at position +1 (12). Furin substrate sequence occurs in many proteins, including the secretory protein stromelysin 3 (10). It has been demonstrated previously that introduction of a 10 amino acid furin substrate sequence of stromelysin 3 into MMP1 (10), sandwiched between the pro and active segments of the enzyme, renders this protein, which is normally secreted in a latent form, to be secreted in an active form. Furin is localized in the trans-Golgi network and catalyzes cleavage of various protein precursors. For example, proinsulin has been engineered to be processed to an active form by introduction of furin substrate sequence in the cleavage sites of the propeptide (7). Similarly, fusion of a secretory protein signal sequence allows expression of the Fc fragment of human IgG1 in COS-1 cells in a secretory form (8). Different vectors have been designed for expression of secretory proteins. Some require co-transfection of a second vector containing a reporter gene (5). Others produce fusion proteins that in many cases impair normal activity of the protein of interest and necessitate laborious proteolytic cleavage protocols after production (9). We describe the design of a new expression vector for secretory proteins that eliminates some of the foregoing difficulties. To illustrate the strategy, we describe experiments using a construct containing bacterial RNase H as a reporter gene fused in-frame to progelatinase A, with a 30-bp furin substrate of stromelysin 3 sandwiched between them. Bacterial RNase H served as reporter gene for the transfection because its detection entails a simple and sensitive assay, and it is not expected to be secreted under normal conditions into the extracellular compartment. Also, the furin substrate sequence insertion between the gene of interest (gelatinase A) and the reporter gene (RNase H) renders the two genes to be secreted in separate forms with no need to further process the gene of interest. Gelatinase A-furin substrate-bacterial RNase H (GFR; Figure 1) was constructed using polymerase chain reaction (PCR) in several steps.